KR102168915B1 - Enzymatic synthesis of L-nucleic acids - Google Patents

Abstract

The present invention relates to a method of adding one or more L-nucleotides to the 3'end of a first L-nucleic acid, wherein the method is in the presence of a protein containing an enzymatic activity exhibiting moiety. The step of reacting the first L-nucleic acid with the one or more L-nucleotides, and the enzymatic activity may add one or more L-nucleotides to the 3'end of the first L-nucleic acid.

Description

Enzymatic synthesis of L-nucleic acids

The present invention is a method of adding one or more L-nucleotides to the 3'end of the first L-nucleic acid, a method of amplifying a target L-nucleic acid, a protein comprising an enzymatic activity exhibiting moiety, Polymerases containing an amino acid sequence whose amino acid sequence is D-amino acid, a polymerase variant of wild-type polymerase consisting of the amino acid sequence of SEQ ID NO: 15, and wild-type polymerase consisting of the amino acid sequence of SEQ ID NO: 1 Polymerase variant of, the use of a protein containing an enzymatic activity exhibiting moiety in the method of adding one or more L-nucleotides, enzymatic activity in a method of amplifying a target L-nucleic acid Use of a protein containing an enzymatic activity exhibiting moiety, a method of identifying a target molecule binding to an L-nucleic acid molecule, a method of producing a polymerase and a protein, respectively, and a first D-peptide or It relates to a method of linking a first D-protein and a second D-peptide or a second D-protein to each other.

In a broad sense, the usefulness of genetic engineering has contributed significantly to progress in the fields of pharmaceuticals and diagnostics as well as basic research over the past decades. The power of synthesis provided by genetic technology goes beyond chemical synthesis. In particular, genetic technology and genetic engineering allow for virtually unlimited production of L-peptide and L protein using enzymatic systems of prokaryotic and eukaryotic cells. Enzymes and polymerases, in particular wild-type or wild-type variants such as these, link building blocks such as D-nucleic acids, i.e., D-nucleotides, to a length that can be achieved by chemical synthesis rather than minimal reasonable yield. Allow D-nucleic acid to be synthesized.

Due to their chiral specificity, enzymes used in genetic technology can only use individual building blocks and substrates, and fit their own chirality. Each building block and substrate of opposite chirality cannot be used for the activity of the enzyme. Further, by the principle of chiral reciprocity, the processing of each building block and substrate of opposite chirality requires enzymes with opposite chirality.

The principle of chiral reciprocity is used intensively in the production of target-binding L-nucleic acids known and referred to as, for example, spiegelmers. To date, Spiegelmers have been identified by the first step, the process of using a D-nucleic acid library for in vitro selection for the enantiomeric form of the target molecule or target structure, such as a D-peptide or D-protein. In the second step, the identified D-nucleic acid bound to the target molecule or enantiomeric form of the target structure is prepared corresponding to the L-nucleic acid. As a result of the chiral reciprocity principle, these L-nucleic acids, i.e. Spiegelmers, can bind to the true or actual target molecule such as L-peptide or L-protein, and the D-peptide or D- They do not bind to their enantiomeric forms such as proteins. Preferably, such a true or actual target molecule or target structure is a target molecule or target structure present in a biological system such as a human or animal body. A method for preparing such a spiegelmer is described in, for example, an'aptamer handbook' (eds. Klussmann, 2006).

One way to make the process of easily identifying Spiegelmers is that L-nucleic acid is directly derived from the L-nucleic acid library using the target molecule or target structure in the corresponding enantiomeric form represented by the true or actual target molecule or target structure. The process can be redesigned to be selected. Since part of the process is the amplification of these L-nucleic acids that initially bind to the target molecule and target structure, respectively, a polymerase that adds one or more nucleotides to the L-primer is required. To date, no polymerase comprising L-amino acids capable of performing as described above is known. For this reason, there is a demand for polymerases and similar enzymes composed of D-amino acids. Since genetic technology cannot provide a functionally activated polymerase consisting of such D-amino acids, chemical synthesis is required. However, the synthesis of D-proteins or D-polypeptides is limited for relatively small molecules. Currently, the largest synthetic D-protein is the D-protein form of vascular endothelial growth factor (abbreviation VEGF-A) consisting of 102 D-amino acids, an angiogenic protein (Mandal et al., 2012), a polymerase, but typically 300. It consists of more than four amino acids.

The basic object of the present invention is to provide a method of adding one or more nucleotides to L-nucleic acid such as a primer. Another basic object of the present invention is to provide a method for amplifying a target L-nucleic acid using nucleotides. Another basic object of the present invention is to provide a means to make this method work.

These and other basic problems of the present invention are solved by the contents of the attached independent claims. Preferred embodiments may be selected from the appended dependent claims.

The basic problem of the present invention is solved by a method of adding at least one L-nucleotide to the 3'end of the first L-nucleic acid in the first aspect, which is also the first embodiment of the first aspect, and the method is enzymatically And reacting the first L-nucleic acid with the one or more L-nucleotides in the presence of a protein containing an enzymatic activity exhibiting moiety, wherein the enzymatic activity is the first L-nucleic acid One or more L-nucleotides may be added to the 3'end of.

In the second embodiment of the first aspect, which is also one embodiment of the first embodiment of the first aspect, the site exhibiting enzymatic activity is composed of an amino acid sequence, and the amino acid of the amino acid sequence is a D-amino acid.

In the third embodiment of the first aspect, which is also one embodiment of the first and second embodiments of the first aspect, the moiety exhibiting enzymatic activity is a polymerase activity exhibiting moiety.

In the fourth embodiment of the first aspect, which is also an embodiment of the first, second and third embodiments of the first aspect, the enzymatic activity is a polymerase activity.

In the fifth embodiment of the first aspect, which is also an embodiment of the fourth embodiment of the first aspect, the polymerase activity is a thermostable polymerase activity.

In the sixth embodiment of the first aspect, which is also one embodiment of the third, fourth and fifth embodiments of the first aspect, the amino acid sequence of the region exhibiting polymerase activity includes at least 300 amino acids.

In the seventh embodiment of the first aspect, which is also one embodiment of the sixth embodiment of the first aspect, the amino acid sequence of the region exhibiting polymerase activity includes 300 to 900 amino acids, and preferably 300 to 600 amino acids. It includes, more preferably 300 to 360 amino acids, and most preferably 340 to 360 amino acids.

In the eighth embodiment of the first aspect, which is also one embodiment of the fourth, fifth, sixth and seventh embodiments of the first aspect, the polymerase activity is a DNA-polymerase activity.

In the ninth embodiment of the first aspect, which is also an embodiment of the eighth embodiment of the first aspect, the DNA-polymerase activity is a DNA-dependent polymerase activity.

In the tenth embodiment of the first aspect, which is also one embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth embodiment of the first aspect, the enzymatic The site showing activity is an enzyme.

In the eleventh embodiment of the first aspect, which is also an embodiment of the third, fourth, fifth, sixth, seventh, eighth and ninth embodiments of the first aspect, the site exhibiting the polymerase activity is polymerization It is an enzyme.

In the twelfth embodiment of the first aspect, which is also one embodiment of the first, second, third, fourth, eighth, ninth, tenth and eleventh embodiment of the first aspect, the polymerase activity is exhibited. Sites are African Swine Fever Virus Polymerase X, Thermus thermophilus polymerase X core domain, rat Polymerase beta, and eukaryotic polymerase. Beta (eukaryotic Polymerase beta), Klenow Fragment, Klenow exo-polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Sequenase, T7 Selected from the group consisting of DNA polymerase, SP6 polymerase, DNA polymerase I, polymerase lambda, and variants thereof,

Preferably, the region exhibiting polymerase activity is African swine fever virus polymerase X or a variant thereof, and is composed of an amino acid sequence selected from an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 4.

A thirteenth implementation of the first aspect that is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh embodiment of the first aspect In an example, the site showing the polymerase activity is DPO4 polymerase, Thermococcus litoralis DNA polymerase, Pyrococcus sp. DNA polymerase, Pyrococcus furiosus DNA Polymerase, Pfuturbo Polymerase, Sulfolobus solfataricus DNA Polymerase, Thermococcus gorgonarius DNA Polymerase, KOD Polymerase, Taq Polymerase, Tth Polymerase, Pyrobest ) Polymerase, Pwo polymerase, Sac polymerase, Bst polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerization Enzyme, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, AmpliTaq, Stoffel fragment, 9°Nm DNA Polymerase, Terminator, Therminator II, Phusion High Fidelity Polymerase, Paq5000, Pfx-50, Proofstart, FideliTaq, Elongase, and variants thereof Is selected from the group,

Preferably, the region exhibiting polymerase activity is Dpo4 polymerase or a variant thereof, and is composed of a selected amino acid sequence of an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 to 22.

The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiment of the first aspect In the fourteenth embodiment of the first aspect, the reaction step comprises at least one L-nucleotide, preferably 5 to 20,000 L-nucleotides, preferably 10 to 2,000 L-nucleotides, and more preferably 50 to the first L-nucleic acid. To 500 L-nucleotides, most preferably 50 to 100 L-nucleotides are added.

One embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiment of the first aspect In the fifteenth embodiment of the first aspect, the addition of one or more L-nucleotides to the first L-nucleic acid is due to covalent bonding of one or more L-nucleotides to the first L-nucleic acid, preferably This is due to the formation of a 3'-5' phosphodiester linkage between the 3'OH of the first L-nucleic acid and one 5'phosphate of one or more L-nucleotides.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th and 15th embodiment of the first aspect In the sixteenth embodiment of the first aspect, which is also an embodiment, the first L-nucleic acid is a primer composed of DNA, RNA, modified DNA, modified RNA, or a combination thereof.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and the 1st aspect of the first aspect In the seventeenth embodiment of the first aspect, which is also one embodiment of the sixteenth embodiment, the first L-nucleic acid consists of L-nucleotides and optionally modified L-nucleotides.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th of the first aspect In the eighteenth embodiment of the first aspect, which is also one embodiment of the 16th and seventeenth embodiments, the first L-nucleic acid consists of L-nucleotides.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th of the first aspect In the 19th embodiment of the first aspect, which is also an embodiment of the 16th, 17th and 18th embodiments, the reaction additionally comprises a second L-nucleic acid, and one molecule of the first L-nucleic acid is It is hybridized with one molecule of 2 L-nucleic acid, and is preferably hybridized through Watson-Crick base pairing.

In the twentieth embodiment of the first aspect, which is also one embodiment of the nineteenth embodiment of the first aspect, the moiety exhibiting polymerase activity synthesizes a third L-nucleic acid complementary to the second L-nucleic acid, and the agent The 3 L-nucleic acid includes the first L-nucleic acid and an L-nucleotide added to the 3'end of the first L-nucleic acid.

The basic problem of the present invention is to amplify a target L-nucleic acid in the presence of a protein including an L-nucleotide and an enzymatic activity exhibiting moiety in the second aspect, which is also the first embodiment of the second aspect. Solved by the method, the enzymatic activity can amplify the target L-nucleic acid.

In the second embodiment of the second aspect, which is also one embodiment of the first embodiment of the second aspect, the site exhibiting enzymatic activity is composed of an amino acid sequence, and the amino acid of the amino acid sequence is a D-amino acid.

In the third embodiment of the second aspect, which is also an embodiment of the first and second embodiments of the second aspect, the moiety exhibiting enzymatic activity is a polymerase activity exhibiting moiety.

In the fourth embodiment of the second aspect, which is also one embodiment of the first, second and third embodiments of the second aspect, the enzymatic activity is a polymerase activity.

In the fifth embodiment of the second aspect, which is also an embodiment of the fourth embodiment of the second aspect, the polymerase activity is a thermostable polymerase activity.

In the sixth embodiment of the second aspect, which is also one embodiment of the third, fourth and fifth embodiments of the second aspect, the amino acid sequence of the region exhibiting polymerase activity includes at least 300 amino acids.

In the seventh embodiment of the second aspect, which is also one embodiment of the sixth embodiment of the second aspect, the amino acid sequence of the region exhibiting polymerase activity includes 300 to 900 amino acids, and preferably 300 to 600 amino acids. It includes, more preferably 300 to 360 amino acids, and most preferably 340 to 360 amino acids.

In the eighth embodiment of the second aspect, which is also one embodiment of the fourth, fifth and sixth embodiments of the second aspect, the polymerase activity is a DNA-polymerase activity.

In the ninth embodiment of the second aspect, which is also one embodiment of the eighth embodiment of the second aspect, the DNA-polymerase activity is a DNA-dependent polymerase activity.

In the tenth embodiment of the second aspect, which is also one embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth embodiment of the second aspect, the enzymatic The site showing activity is an enzyme.

In the 11th embodiment of the second aspect, which is also an embodiment of the 3rd, 4th, 5th, 6th, 7th, 8th and 9th embodiments of the second aspect, the site exhibiting polymerase activity is polymerization It is an enzyme.

In the twelfth embodiment of the second aspect, which is also one embodiment of the first, second, third, fourth, eighth, ninth, tenth and eleventh embodiment of the second aspect, the polymerase activity is exhibited. Sites are African Swine Fever Virus Polymerase X, Thermus thermophilus polymerase X core domain, rat Polymerase beta, and eukaryotic polymerase. Beta (eukaryotic Polymerase beta), Klenow Fragment, Klenow exo-polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Sequenase, T7 Selected from the group consisting of DNA polymerase, SP6 polymerase, DNA polymerase I, polymerase lambda, and variants thereof,

Preferably, the region exhibiting polymerase activity is African swine fever virus polymerase X or a variant thereof, and is composed of an amino acid sequence selected from an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 4.

A thirteenth implementation of the second aspect that is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh embodiment of the second aspect In an example, the site showing the polymerase activity is DPO4 polymerase, Thermococcus litoralis DNA polymerase, Pyrococcus sp. DNA polymerase, Pyrococcus furiosus DNA Polymerase, Pfuturbo Polymerase, Sulfolobus solfataricus DNA Polymerase, Thermococcus gorgonarius DNA Polymerase, KOD Polymerase, Taq Polymerase, Tth Polymerase, Pyrobest ) Polymerase, Pwo polymerase, Sac polymerase, Bst polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerization Enzyme, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, AmpliTaq, Stoffel fragment, 9°Nm DNA Polymerase, Terminator, Therminator II, Phusion High Fidelity Polymerase, Paq5000, Pfx-50, Proofstart, FideliTaq, Elongase, and variants thereof Is selected from the group,

Preferably, the region exhibiting polymerase activity is Dpo4 polymerase or a variant thereof, and is composed of a selected amino acid sequence of an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 to 22.

The first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiment of the second aspect In the fourteenth embodiment of the second aspect, the reaction step is carried out under conditions in which the target L-nucleic acid is amplified.

One embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiment of the second aspect In a fifteenth embodiment of the second aspect, the method uses one or more primers, preferably two primers, wherein the one or more primers consist of L-nucleotides and optionally modified L-nucleotides. .

In the sixteenth embodiment of the second aspect, which is also one embodiment of the fifteenth embodiment of the second aspect, the primer consists of L-nucleotides.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and the 2nd aspect In the seventeenth embodiment of the second aspect, which is also one embodiment of the 16th embodiment, the target L-nucleic acid consists of L-nucleotides.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th of the second aspect In the eighteenth embodiment of the second aspect, which is also one embodiment of the 16th and 17th embodiments, the method is a polymerase chain reaction (PCR).

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th of the second aspect In the 19th embodiment of the second aspect, which is also one embodiment of the 16th, 17th and 18th embodiments, the target L-nucleic acid consists of L-DNA.

1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th of the second aspect In the tenth embodiment of the second aspect, which is also one embodiment of the 16th, 17th, 18th and 19th embodiments, the target L-nucleic acid is 20 to 20,000 L-nucleotides, preferably 30 to 2,000 L-nucleotides. , More preferably 40 to 500 L-nucleotides, most preferably 50 to 100 L-nucleotides.

The basic problem of the present invention is solved by a protein containing an enzymatic activity exhibiting moiety in the third aspect, which is also the first embodiment of the third aspect, and the region exhibiting enzymatic activity is an amino acid It consists of a sequence, the amino acid of the amino acid sequence is a D-amino acid, and the enzymatic activity may add one or more L-nucleotides to the 3'end of the first L-nucleic acid.

In the second embodiment of the third aspect, which is also one embodiment of the first embodiment of the third aspect, the moiety exhibiting enzymatic activity is a polymerase activity exhibiting moiety.

In the third embodiment of the third aspect, which is also one embodiment of the first embodiment of the third aspect, the enzymatic activity is a polymerase activity.

In the fourth embodiment of the third aspect, which is also an embodiment of the third embodiment of the third aspect, the polymerase activity is a thermostable polymerase activity.

In the fifth embodiment of the third aspect, which is also an embodiment of the second, third and fourth embodiments of the third aspect, the amino acid sequence of the region exhibiting polymerase activity includes at least 300 amino acids.

In the sixth embodiment of the third aspect, which is also one embodiment of the fifth embodiment of the third aspect, the amino acid sequence of the region exhibiting polymerase activity includes 300 to 900 amino acids, and preferably 300 to 600 amino acids. It includes, more preferably 300 to 360 amino acids, and most preferably 340 to 360 amino acids.

In the seventh embodiment of the third aspect, which is also one embodiment of the third, fourth, fifth and sixth embodiments of the third aspect, the polymerase activity is a DNA-polymerase activity.

In the eighth embodiment of the third aspect, which is also one embodiment of the seventh embodiment of the third aspect, the DNA-polymerase activity is a DNA-dependent polymerase activity.

In the ninth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth embodiment of the third aspect, the enzymatic activity is exhibited. The site is an enzyme.

In the tenth embodiment of the third aspect, which is also an embodiment of the second, third, fourth, fifth, sixth, seventh and eighth embodiments of the third aspect, the moiety exhibiting the polymerase activity is polymerization It is an enzyme.

In the eleventh embodiment of the third aspect, which is also an embodiment of the first, second, third, seventh, eighth, ninth and tenth embodiments of the third aspect, the region exhibiting polymerase activity is Africa African Swine Fever Virus Polymerase X, Thermus thermophilus polymerase X core domain, rat Polymerase beta, eukaryotic polymerase beta Polymerase beta), Klenow Fragment, Klenow exo-polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Sequenase, T7 DNA polymerase , SP6 polymerase, DNA polymerase I, polymerase lambda (polymerase lambda), and is selected from the group consisting of variants (variants) thereof,

Preferably, the region exhibiting polymerase activity is African swine fever virus polymerase X or a variant thereof, and is composed of an amino acid sequence selected from an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 4.

In the twelfth embodiment of the third aspect, which is also one embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth embodiment of the third aspect, The site showing the polymerase activity is DPO4 polymerase, Thermococcus litoralis DNA polymerase, Pyrococcus sp. DNA polymerase, Pyrococcus furiosus DNA polymerase, Pfuturbo polymerase, Sulfolobus solfataricus DNA polymerase, Thermococcus gorgonarius DNA polymerase, KOD polymerase, Taq polymerase, Tth polymerase, Pyrobest polymerase , Pwo polymerase, Sac polymerase, Bst polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, AmpliTaq, Stoffel fragment, 9°Nm Selected from the group consisting of DNA polymerase, Therminator, Therminator II, Phusion High Fidelity polymerase, Paq5000, Pfx-50, Proofstart, FideliTaq, Elongase, and variants thereof And

Preferably, the region exhibiting polymerase activity is Dpo4 polymerase or a variant thereof, and is composed of a selected amino acid sequence of an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 to 22.

The basic problem of the present invention is solved by a polymerase comprising the amino acid sequence of SEQ ID NO: 15 in the fourth aspect, which is also the first embodiment of the fourth aspect, and the amino acid of the amino acid sequence is a D-amino acid.

The basic problem of the present invention is solved by a polymerase containing the amino acid sequence of SEQ ID NO: 1 in the fifth aspect, which is also the first embodiment of the fifth aspect, and the amino acid of the amino acid sequence is a D-amino acid.

The basic problem of the present invention is solved by a polymerase variant of wild-type polymerase in the sixth aspect, which is also the first embodiment of the sixth aspect, and the wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15, The polymerase variant has a polymerase activity, preferably a thermostable polymerase activity.

In the second embodiment of the sixth aspect, which is also one embodiment of the first embodiment of the sixth aspect, the polymerase variant comprises an amino acid sequence, and the amino acid sequence of the polymerase variant is at least one amino acid position. Is different from the amino acid sequence of wild-type polymerase.

In the third embodiment of the sixth aspect, which is also one embodiment of the first and second embodiments of the sixth aspect, the amino acid sequence of the polymerase variant is different from the amino acid sequence of the wild-type polymerase in one or two amino acid positions. Do.

In the fourth embodiment of the sixth aspect, which is also one embodiment of the first, second and third embodiments of the sixth aspect, the amino acid sequence of the polymerase variant is 155 and/or 203th of the amino acid sequence of SEQ ID NO: 15. The amino acid position or the corresponding amino acid position is different from the amino acid sequence of the wild-type polymerase, and preferably the 155 and/or 203th amino acid positions of the amino acid sequence are substituted with cysteine.

In the fifth embodiment of the sixth aspect, which is also one embodiment of the first, second, third and fourth embodiments of the sixth aspect, the polymerase variant is selected from the group consisting of amino acid sequences of SEQ ID NOs: 16 to 18. It consists of an amino acid sequence.

In the sixth embodiment of the sixth aspect, which is also one embodiment of the first, second and third embodiments of the sixth aspect, the polymerase variant is an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 19 to 22. Is composed.

In the seventh embodiment of the sixth aspect, which is also one embodiment of the first, second, third, fourth, fifth and sixth embodiments of the sixth aspect, the amino acid of the amino acid sequence of the polymerase variant is D- It is an amino acid.

In the eighth embodiment of the sixth aspect, which is also one embodiment of the first, second, third, fourth, fifth and sixth embodiments of the sixth aspect, the amino acid of the amino acid sequence of the polymerase variant is L- It is an amino acid.

The basic problem of the present invention is solved by a polymerase variant of the wild-type polymerase in the seventh aspect, which is also the first embodiment of the seventh aspect, and the wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 1, The polymerase variant has polymerase activity.

In the second embodiment of the seventh aspect, which is also one embodiment of the first embodiment of the seventh aspect, the polymerase variant comprises an amino acid sequence, and the amino acid sequence of the polymerase variant is at least one amino acid position. Is different from the amino acid sequence of wild-type polymerase.

In the third embodiment of the seventh aspect, which is also one embodiment of the first and second embodiments of the seventh aspect, the amino acid sequence of the polymerase variant differs from the amino acid sequence of the wild-type polymerase by one amino acid position.

In the fourth embodiment of the seventh aspect, which is also one embodiment of the first, second and third embodiments of the seventh aspect, the amino acid sequence of the polymerase variant is at least one amino acid position of the wild-type polymerase. Different from the amino acid sequence, the one or more amino acid positions are selected from the 80, 86, and 124th amino acid positions of the amino acid sequence of SEQ ID NO: 1 or amino acid positions corresponding thereto.

In the fifth embodiment of the seventh aspect, which is also one embodiment of the first, second, third and fourth embodiments of the seventh aspect, the polymerase variant is selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 to 4. It consists of an amino acid sequence.

In the sixth embodiment of the seventh aspect, which is also one embodiment of the first, second, third, fourth and fifth embodiments of the seventh aspect, the amino acid of the amino acid sequence of the polymerase variant is a D-amino acid.

1st, 2nd, 3rd, 4th of the 7th aspect. In the seventh embodiment of the seventh aspect, which is also one embodiment of the fifth and sixth embodiments, the amino acid of the amino acid sequence of the polymerase variant is an L-amino acid.

The basic problem of the present invention is that in the method of adding one or more L-nucleotides to the 3'end of L-nucleic acid in the eighth aspect, which is also the first embodiment of the eighth aspect, the enzymatic activity exhibiting moiety ) Is solved by the use of a protein containing.

The basic object of the present invention is to include an enzymatic activity exhibiting moiety in the method of amplifying a target L-nucleic acid in the presence of L-nucleotides in the ninth aspect, which is also the first embodiment of the ninth aspect. It is solved by the use of the protein.

In the second embodiment of the ninth aspect, which is also an embodiment of the first embodiment of the ninth aspect, the method of amplifying the target L-nucleic acid is a polymerase chain reaction (PCR).

In the second embodiment of the eighth aspect and the third embodiment of the ninth aspect, which is also an embodiment of the first embodiment of the eighth aspect, the protein is a protein of any one of the third to seventh aspects, and the Proteins are composed of an amino acid sequence, and the amino acids in the amino acid sequence are D-amino acids.

The basic object of the present invention is a method of identifying a target molecule binding to an L-nucleic acid molecule comprising the following steps in a tenth aspect, which is also the first embodiment of the tenth aspect,

(a) forming a heterogeneous population of L-nucleic acid molecules;

(b) contacting the target molecule with the heterogeneous population of L-nucleic acid molecules in step (a);

(c) separating the L-nucleic acid molecule not bound to the target molecule; And

(d) amplifying the L-nucleic acid molecule bound to the target molecule, wherein the amplifying step uses the protein of any one embodiment of the third to seventh aspects,

The protein is composed of an amino acid sequence, and the amino acid in the amino acid sequence is resolved by a method in which the amino acid is D-amino acid.

In the second embodiment of the tenth aspect, which is also an embodiment of the first embodiment of the tenth aspect, the method comprises:

(e) sequencing the L-nucleic acid molecule bound to the target molecule; And

(f) synthesizing a nucleic acid molecule having the same nucleotide sequence as the nucleotide sequence of the sequenced L-nucleic acid molecule in the step (e).

In the third embodiment of the tenth aspect, which is also one embodiment of the first and second embodiments of the tenth aspect, the nucleic acid molecules of the heterogeneous population of L-nucleic acid molecules of step (a) are at their 5'end and 3' Each terminal includes a primer binding site, and a sequence complementary to the primer binding site so that the L-nucleic acid molecule obtained in step (d) is amplified by a polymerase chain reaction, The polymerase used in the polymerase chain reaction is a protein of any one of the third to seventh aspects, and the primer used in the polymerase chain reaction is composed of L-nucleotides, and is used in the polymerase chain reaction. Nucleotides are L-nucleotides.

In the fourth embodiment of the tenth aspect, which is also one embodiment of the first, second and third embodiments of the tenth aspect, after the step (d)

(da) a step of contacting the amplified nucleic acid molecule with a target molecule is introduced,

Step (b) and optionally step (c) and/or step (d) are performed before step (e), and step (da), step (b), step (c) and optionally step (d) The steps are performed once or several times in sequence.

In the fifth embodiment of the tenth aspect, which is also one embodiment of the first, second, third and fourth embodiments of the tenth aspect, the target molecule that binds to the L-nucleic acid is DNA.

In the sixth embodiment of the tenth aspect, which is also an embodiment of the first, second, third, fourth and fifth embodiments of the tenth aspect, the target molecule binding to the L-nucleic acid molecule is an L-nucleotide. Is composed.

The basic object of the present invention is a method for producing a protein of any one of the 3rd to 7th embodiments, comprising the following steps in the 11th aspect, which is also the first embodiment of the 11th aspect,

a) At least one fragment of the protein of any one of the third to seventh embodiments is chemically synthesized, and the entire fragment forms the amino acid sequence of the protein, preferably the fragment is solid-phase peptide synthesis ( synthesized by solid phase peptide synthesis); And

b) the fragments of step a) are linked to each other by segment condensation, native chemical ligation, enzymatic ligation, or a combination thereof,

The protein is composed of an amino acid sequence, and the amino acid in the amino acid sequence is solved by a method wherein the amino acid is D-amino acid.

In the second embodiment of the eleventh aspect, which is also an embodiment of the first embodiment of the eleventh aspect, the enzyme used for the enzymatic linkage is Clostripain.

The basic task of the present invention is to connect the first D-peptide or the first D-protein and the second D-peptide or the second D-protein by enzymatic linkage in the twelfth aspect, which is also the first embodiment of the twelfth aspect. It is solved by the method

The first D-peptide or the first D-protein is protected by a protection group at its N-terminus, and a 4-guanidinophenyl ester group at its C-terminus (4- guanidinophenylester group),

The second D-peptide or the second D-protein includes a free N-terminus and a thioalkylester group or a thioarylester group at its C-terminus.

In the second embodiment of the twelfth aspect, which is also one embodiment of the first embodiment of the twelfth aspect, the enzyme used for the enzymatic linkage is Clostripain.

The inventors have surprisingly found that proteins consisting of functionally activated D-amino acids can be chemically synthesized, whereby these proteins have a typical size displayed by polymerases. More specifically, the present inventors have identified a method for synthesizing D-protein and D-polymerase (eg, a polymerase composed of D-amino acids activated as a polymerase). Based on this fact, the proteins and enzymatic activities required for the enzymatic synthesis of L-nucleic acid and L-nucleic acid molecules are now available. This enzymatic synthesis of L-nucleic acid and L-nucleic acid molecules involves the addition of one or more L-nucleotides to the 3'end of the first L-nucleic acid and targeting L-nucleic acids in the presence of L-nucleotides as L-nucleic acids. A method of amplifying, ie, a method in which the amplification product is L-nucleic acid, is included, but is not limited thereto.

Since these methods and enzymatic activity are part of an alternative process for identifying spiegelmers, they can currently be put into practice as an alternative process for identifying spiegelmers.

The present inventors have developed a method of adding one or more L-nucleotides to the 3'end of the first L-nucleic acid, and the method is in the presence of a protein containing an enzymatic activity exhibiting moiety. And reacting the first L-nucleic acid with the one or more L-nucleotides, and the enzymatic activity may add one or more L-nucleotides to the 3'end of the first L-nucleic acid.

According to a preferred embodiment of the present invention, the enzymatic activity is 5 to 20,000 L-nucleotides, preferably 10 to 2,000 L-nucleotides, more preferably 50 to 500 L-nucleotides at the 3'end of the first L-nucleic acid. L-nucleotides, most preferably 50 to 100 L-nucleotides can be added.

Preferably, the term addition as used herein refers to a covalent bond between molecules, and in the present invention, the covalent bond of one or more L-nucleotides to the first L-nucleic acid, preferably 3'OH of the first L-nucleic acid. And the formation of a 3'-5' phosphodiester linkage between one 5'phosphate of one or more L-nucleotides. According to the present invention, the L-nucleotide added to the L-nucleic acid forms the 3'end of the L-nucleic acid extended by the L-nucleotide.

According to a preferred embodiment of the present invention, the method of adding one or more L-nucleotides to the 3'end of the first L-nucleic acid of the present invention includes a second L-nucleic acid, and one of the first L-nucleic acids The two molecules are hybridized with one molecule of the second L-nucleic acid, and are preferably hybridized through Watson-Crick base pairing. According to a more preferred embodiment of the present invention, the method synthesizes a third L-nucleic acid complementary to the second L-nucleic acid, and the third L-nucleic acid is the first L-nucleic acid and the first L- It includes an L-nucleotide added to the 3'end of the nucleic acid. That is, with respect to the first L-nucleic acid, one or more L-nucleotides are added to the 3'end of the first L-nucleic acid to become the third L-nucleic acid.

The protein comprising a site exhibiting enzymatic activity of the present invention includes another residue or part of the protein that does not have enzymatic activity. The protein comprising a site exhibiting enzymatic activity of the present invention includes a protein having a site exhibiting enzymatic activity alone and a protein having a site exhibiting enzymatic activity and other residues or parts, and other residues of the protein or Some do not have any enzymatic activity. The amino acid sequence of the region exhibiting enzymatic activity of the present invention includes 300 to 900 amino acids, preferably 300 to 600 amino acids, more preferably 300 to 360 amino acids, and most preferably 340 To 360 amino acids.

The protein comprising the site exhibiting enzymatic activity of the present invention is preferably a site exhibiting polymerase activity. The protein containing the site exhibiting polymerase activity of the present invention includes a polymerase having a site exhibiting polymerase activity alone and a polymerase having a site exhibiting polymerase activity and another moiety or part, the polymerase The other moieties or portions of do not have any polymerase activity. The amino acid sequence of the region exhibiting the polymerase activity of the present invention contains 300 to 900 amino acids, preferably 300 to 600 amino acids, more preferably 300 to 360 amino acids, and most preferably 340 To 360 amino acids.

The site exhibiting polymerase activity of the present invention is preferably a site exhibiting thermostable polymerase activity, more preferably a site exhibiting thermostable DNA-polymerase activity, and most preferably thermostable DNA-dependent DNA- It is a site showing polymerase activity.

The site exhibiting polymerase activity of the present invention is preferably a site exhibiting DNA-polymerase activity, more preferably a site exhibiting DNA-dependent DNA-polymerase activity or a site exhibiting thermostable DNA-polymerase activity. , Most preferably, it is a site exhibiting thermostable DNA-dependent DNA-polymerase activity.

The term enzymatic activity as used herein is a catalysis of a specific reaction, preferably the addition of one or more nucleotides to the 3'end of the nucleic acid, amplification of the nucleic acid and/or polymerase activity, more preferably L- It is the addition of one or more L-nucleotides to the 3'end of the nucleic acid and amplification of the L-nucleic acid.

The term polymerase activity according to the present invention is an enzyme capable of polymerizing L-nucleotides and/or polymerizing L-nucleotides to L-nucleic acid, preferably, the L-nucleotide is L-nucleoside triphosphate. (L-nucleoside triphosphates).

The polymerase activity of the present invention is preferably a thermostable polymerase activity, more preferably a thermostable DNA-polymerase activity, and most preferably a thermostable DNA-dependent DNA-polymerase activity.

The polymerase activity of the present invention is preferably a DNA-polymerase activity, more preferably a DNA-dependent DNA-polymerase activity or a thermostable DNA-polymerase activity, and most preferably a thermostable DNA-dependent DNA- It is polymerase activity.

Known polymerases are natural resources or optimized or variant variants of polymerases from natural resources. The polymerase is composed of chiral building blocks, ie L-amino acids. Thus, the structure of the polymerase is chiral in nature and leads to the recognition of stereospecific substrates. Thus, these enzymes only accept substrate molecules that are suitable, ie corresponding to a chiral configuration. Known polymerases polymerize D-nucleotides or D-nucleoside triphosphates, which synthesize complementary D-nucleic acid strands composed of D-nucleotides using D-nucleic acid composed of D-nucleotides as a template strand. In addition to the template strand, the polymerase hybridizes to the template strand and selectively uses a primer composed of D-nucleotides. Since naturally occurring nucleic acids are composed of D-nucleotides, they can be processed, e.g., amplified, by proteins and enzymes, especially proteins and enzymes composed of D-amino acids, and L-nucleic acids are converted to D-amino acids. It is not recognized by each of the proteins and enzymes constructed. Therefore, the target molecule or L-nucleic acid that binds to the target structure is also called spingegelmer, and cannot be obtained directly by the in vitro selection process using the natural occurrence from the target molecule or target structure.

The present inventors have surprisingly found that it is possible to produce a polymerase capable of adding L-nucleic acid nucleotides to a primer composed of L-nucleotides hybridizing to an L-nucleic acid template strand. In addition, the present inventors have surprisingly found that it is possible to produce a polymerase that can be used for the amplification of L-nucleic acid, preferably in a process known as polymerase chain reaction (abbreviation PCR).

Polymerase is an enzyme that polymerizes nucleoside triphosphate. Polymerase uses a template nucleic acid strand to synthesize a nucleic acid strand complementary to the template nucleic acid strand. In addition to the template nucleic acid strand, the polymerase selectively uses a primer that hybridizes based on a base complementary to the template nucleic acid strand. The template nucleic acid strand, the primer, and the nucleic acid strand synthesized by the polymerase may independently be DNA or RNA. Preferably the polymerase used in the present invention includes a DNA polymerase and an RNA-polymerase, and preferably, a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase such as a reverse transcriptase, an RNA-dependent RNA polymerase, and It is an RNA-dependent DNA polymerase. More preferably, the polymerase is a thermostable polymerase. The polymerase need not contain all of the amino acids found in the natural or wild-type enzyme, but should be sufficient for the polymerase to achieve the desired catalytic activity. In an embodiment, the polymerase activity is, for example, a group comprising catalytic activities including 5'-3' polymerization, 5'-3' exonuclease and 3'-5' exonuclease activity It is a catalytic activity selected from

The polymerase of the present invention is composed of D-nucleic acid, and polymerizes L-nucleotide or L-nucleoside triphosphate, and the polymerase of the present invention uses L-nucleic acid composed of L-nucleotide as a template strand. -Synthesize complementary L-nucleic acid strands composed of nucleotides. In addition to the above template strand, the polymerase of the present invention is hybridized to the template strand and selectively uses a primer composed of L-nucleotides. The template strand, the primer, and the synthesized nucleic acid strand may independently be L-DNA or L-RNA. The polymerase of the present invention includes a DNA polymerase composed of D-amino acids and an RNA-polymerase composed of D-amino acids, preferably a DNA-dependent DNA polymerase composed of D-amino acids, and a reverse transcriptase composed of D-amino acids. Such as RNA-dependent DNA polymerase, RNA-dependent RNA polymerase composed of D-amino acids, and RNA-dependent DNA polymerase composed of D-amino acids. More preferably, the polymerase of the present invention is a thermostable polymerase composed of D-amino acids. The polymerase of the present invention need not contain all amino acids found in natural enzymes, but should be sufficient for the polymerase of the present invention to perform the desired catalytic activity. Catalytic activities include, for example, 5'-3' polymerization, 5'-3' exonuclease and 3'-5' exonuclease activity.

In the present specification, a polymerase consisting solely of L-amino acids is preferably referred to as'all-L polymerase'.

In the present specification, a polymerase consisting solely of D-amino acids is preferably referred to as'all-D polymerase'.

The polymerase of a preferred embodiment of the present invention is African Swine Fever Virus Polymerase X (African Swine Fever Virus Polymerase X), Thermus thermophilus polymerase X core domain (Thermus thermophilus polymerase X core domain), rat polymerase beta (rat Polymerase beta), eukaryotic polymerase beta, Klenow Fragment, Klenow exo-polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Sequenase, T7 DNA polymerase, SP6 polymerase, DNA polymerase I, polymerase lambda, DPO4 polymerase, Thermococcus litoralis DNA polymerase, Pyrococcus species sp.) DNA polymerase, Pyrococcus furiosus DNA polymerase, Pfuturbo polymerase, Sulfolobus solfataricus DNA polymerase, Thermococcus gorgonarius DNA polymerase , KOD polymerase, Taq polymerase, Tth polymerase, Pyrobest polymerase, Pwo polymerase, Sac polymerase, Bst polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 Polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, AmpliTaq, Stoffel fragment, 9°Nm DNA polymerase, terminator (Therm inator), Terminator II, Phusion High Fidelity polymerase, Paq5000, Pfx-50, Proofstart, FideliTaq, Elongase, and variants thereof.

The polymerase of a more preferred embodiment of the present invention is African porcine fever virus polymerase X consisting of the amino acid sequence of SEQ ID NO: 1. The polymerase of a more preferred embodiment of the present invention is a variant of African swine fever virus polymerase X, most preferably African swine fever virus of an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2 to 4 It is a variant of polymerase X.

The polymerase of a more preferred embodiment of the present invention is Dpo4 polymerase consisting of the amino acid sequence of SEQ ID NO: 15. The polymerase of a more preferred embodiment of the present invention is a variant of Dpo4 polymerase, most preferably a variant of Dpo4 polymerase consisting of an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 16 to 22.

A variant of a polymerase is a polymerase whose one or more amino acid positions differ from the amino acid sequence of the polymerase. Preferably, the position of the amino acid in the amino acid sequence is determined by its position relative to the N-terminus and C-terminus of the polymerase and/or its position relative to the amino acid surrounding the amino acid, thereby

a) when the polymerase is truncated at the N-terminus (truncated), the position of the amino acid is determined by its position relative to the C-terminus of the polymerase and its position relative to the amino acid surrounding the amino acid,

b) when the polymerase is truncated at the C-terminus (truncated), the position of the amino acid is determined by its position relative to the N-terminus of the polymerase and its position relative to the amino acid surrounding the amino acid, and

b) When the polymerase is truncated at the N-terminus and C-terminus, the position of the amino acid is determined by its position relative to the amino acid surrounding the amino acid.

The polymerase of the present invention is thermally stable, which is relatively unaffected by rising temperatures. In one specific, non-limiting example, the polymerase having heat stability is at a temperature of at least 50°C, such as 50°C, 60°C, 75°C, 80°C, 82°C, 85°C, 88°C, 90°C, 92°C , Not affected by 95°C or higher temperatures.

In the course of polymerization of L-nucleoside triphosphate, the polymerase adds one nucleoside triphosphate to another triphosphate, preferably resulting in an oligonucleotide, which is also referred to as a nucleic acid. In a preferred embodiment, the polymerase adds one nucleoside triphosphate to one nucleoside triphosphate or to the terminal nucleotide of the nucleic acid, for example, the nucleotide is a chain terminator, such as a dideoxynucleotide. terminator) in the case of nucleotides. Such chain terminator nucleotides are used for nucleic acid sequencing and are known to those of skill in the art.

The polymerization process of L-nucleoside triphosphate can be used for amplification of L-nucleic acid, preferably target L-nucleic acid.

Amplification can be any process that increases the number of copies of a nucleic acid, preferably a target L-nucleic acid.

In a preferred embodiment, the target L-nucleic acid is 20 to 20,000 L-nucleotides, preferably 30 to 2,000 L-nucleotides, more preferably 40 to 500 L-nucleotides, most preferably 50 to 100 L-nucleotides. Is composed.

An example of amplification is the contact of a nucleic acid with a pair of primers under conditions in which primers hybridize to a nucleic acid template. The primers are extended by polymerase by addition of one or more nucleoside triphosphates to the primers under suitable conditions, separated from the nucleic acid template, and then re-annealed, extended and separated to amplify the copy number of the nucleic acid molecule.

In vitro amplification products can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation and/or nucleic acid sequencing using standard techniques.

Alternative in vitro amplification techniques include transcription-free isothermal amplification, strand displacement amplification, and NASBA™ RNA transcription-free amplification, and are known to those of skill in the art.

Some amplification methods rely on enzymatic replication of nucleic acids and thermal cycling consisting of repeated heating and cooling cycles of the reaction for melting of nucleic acids, preferably double-stranded nucleic acids. This temperature repetition step first requires physical separation of the two strands of double-stranded nucleic acid at a high temperature in a process called nucleic acid melting. Then, at a low temperature, each strand is used as a template in nucleic acid synthesis by polymerase, and the target nucleic acid is selectively amplified. A primer containing a sequence complementary to a target region along with a polymerase is a key element enabling selection and repetitive amplification (the method is named below). Since the amplification method is based on a temperature repetition process, the generated nucleic acid itself is used as a template for replication, and the nucleic acid template is amplified exponentially while a chain reaction is performed.

The most prominent amplification method by temperature amplification is polymerase chain reaction (abbreviation. PCR).

The primer is a short nucleic acid molecule composed of DNA or RNA or a combination thereof, and preferably, the length of the primer is a DNA oligonucleotide of 10 nucleotides or more. More preferably, the length of the primer may be about 15, 20 or 25 nucleotides or longer. The primer is annealed to the complementary target nucleic acid strand by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid sequence, and then the primer is extended along the target nucleic acid strand by a polymerase. Primer pairs can be used for amplification of nucleic acids, such as PCR or other nucleic acid amplification methods known in the art.

The use of the polymerase of the present invention composed of D-amino acid requires that the primer and the complementary target nucleic acid strand be composed of L-nucleotides. Preferably at least one primer consists of an L-nucleotide and an optionally modified L-nucleotide.

Methods of making and using nucleic acid primers and probes are described in literature such as Sambrock et al. (1989). PCR primer pairs can be derived from known sequences, e.g., using a computer program for that purpose, such as a primer. Those of skill in the art will understand that the specificity of a particular probe or primer increases with length.

The polymerase of the present invention is composed of D-amino acids. Since the polymerase of the present invention is composed of D-amino acids, it cannot be isolated from natural sources, cannot be produced by recombinant expression using bacteria, yeast, fungi, viruses or animal cells, and must be prepared by chemical processes, Preferably, it is a combination of a linking method and solid-phase peptide synthesis (abbreviation. SPPS).

Solid-phase peptide synthesis is the synthesis of a peptide or fragment of a state-of-the-art protein, and a peptide chain can be constructed by treating small solid beads and insoluble porous materials with a functional group ('linker'). The peptide will remain covalently attached to the beads until cleaved by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. Thus, the peptide can be'immobilized' in this solid phase, flushing out liquid reagents and synthetic by-products while remaining during the filtration process. The general principle of SPPS is one of the repeated cycles of coupling-wash-deprotection-wash. The free N-terminal amine of the peptide linked to the solid phase is coupled to a single N-protected amino acid unit (see below). These units are deprotected and additional amino acids are attached as new N-terminal amines are revealed. The excellence of this technique lies in part in its ability to perform washing cycles after each reaction, and removes excess reagent with all the increasing peptides of interest remaining covalently attached to the insoluble resin. There are two types of SPPS that are mainly used-Fmoc and BOC. The N-terminus of the amino acid monomer is protected by either group, so it is added to the unprotected amino acid chain. SPPS is limited by yield, and peptides and proteins in the range of generally 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulties are also sequence dependent. Large synthetic oligopeptides and proteins can be accessed using linkage methods such as fragment condensation, natural chemical linkage or enzymatic linkage so that the two peptides can be coupled together. However, the largest D-protein synthesized so far is the D-protein form of vascular endothelial growth factor (abbreviation VEGF-A) consisting of 102 D-amino acids, an angiogenic protein (Mandal et al., 2012), and fragment condensation is a peptide. And the side chains of the amino acids of the peptide are completely protected with chemical groups, and the peptide is coupled in solution.

Natural chemical linkages are carried out in aqueous solutions. The challenge is the preparation of the essential unprotected peptide-thioester building blocks. In natural chemical linkage, the thiolate group of the N-terminal cysteine residue of unprotected peptide 2 in an aqueous buffer at pH 7.0, 20° C.<T<37° C. attacks the C-terminal thioester of the second unprotected peptide 1. This reversible transthioesterification step is chemically selective and regioselective, resulting in the formation of a thioester intermediate 3. The intermediate is rearranged by an intramolecular S,N- acyl shift resulting in the formation of the natural amide ('peptide') bond 4 at the linkage site.

As shown in the examples, the present inventions surprisingly showed that the C-terminal thioester essential for natural chemical linkage is stable under the conditions of enzymatic linkage, whereby natural chemical linkage and enzymatic linkage can be used in combination.

The enzymatic linkage of the D-peptide works using a protease comprising the following steps: (a) preparing an amino component, wherein the amino component is a native D-peptide, and (b) preparing a carboxy component As, the carboxy component includes a leaving group, the amino component is a unique D-peptide, and (c) reacting the amino component with the carboxy component, reacting in the presence of a protease to form a leaving group Cleavage forms a peptide bond between the amino component and the carboxy component to give the native D-polypeptide (see WO2003047743). Preferably the protease is Clostripine.

In addition, the polymerase of the present invention comprises a polymerase that is essentially homologous to the polymerase of the present invention and the specific sequence(s) disclosed herein. The term practical homology will be understood to be at least 75%, preferably 85%, more preferably 90%, most preferably 95%, 96%, 97%, 98% or 99% homology.

In addition, the moiety exhibiting polymerase activity of the present invention includes a polymerase that is essentially homologous to the moiety exhibiting polymerase activity of the present invention and the specific sequence(s) disclosed herein. The term practical homology will be understood to be at least 75%, preferably 85%, more preferably 90%, most preferably 95%, 96%, 97%, 98% or 99% homology.

The actual percentage of homologous amino acids in the polymerase of the present invention or the site exhibiting polymerase activity of the present invention will depend on the total number of amino acids in the polymerase or the site exhibiting polymerase activity. The percent change may be based on the total number of amino acids in the polymerase or the site exhibiting polymerase activity.

As known to those skilled in the art, homology between two polymerases or sites exhibiting two polymerase activities can be determined. More specifically, a sequence comparison algorithm can be used to calculate the percent sequence homology for the test sequence(s) relative to the reference sequence, based on specified program parameters. The test sequence is preferably a polymerase or a site exhibiting polymerase activity that has been tested for homology or homology, whereby a site exhibiting a different polymerase or polymerase activity is also referred to as a homologous reference sequence. The optimal alignment of the amino acid sequence of the polymerase for comparison is, for example, a local homology algorithm of Smith & Waterman (Smith & Waterman, 1981), and a homology alignment algorithm of Needleman & Wunsch (Needleman & Wunsch, 1970). , Pearson & Lipman (Pearson & Lipman, 1988), a search for similarity methods, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis) .) Or it can be done by visual inspection.

An example of an algorithm suitable for determining percent sequence identity is a basic local alignment search tool ("BLAST"), such as in Altschul et al. (Altschul et al. 1990 and Altschul et al, 1997). This is the algorithm used. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information ("NCBI"). The default parameters used to determine sequence identity using software available from NCBI, such as BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences) are described in McGinnis et al. (McGinnis et al., 2004). .

In addition, the polymerase of the present invention includes a polymerase having some degree of relative identity to the polymerase of the present invention, and in particular, a specific polymerase of the present invention disclosed herein and defined by its amino acid sequence. More preferably, the present invention is relative to the polymerase of the present invention at least 75%, preferably 85%, more preferably 90%, most preferably 95% or more, 96%, 97%, 98% or 99 Polymerases having percent identity and in particular certain polymerases of the present invention disclosed herein and defined by their amino acid sequence or portions thereof.

In addition, the moiety exhibiting polymerase activity of the present invention is a moiety exhibiting polymerase activity having a certain degree of relative identity to the moiety exhibiting polymerase activity of the present invention, and in particular disclosed herein and defined by its amino acid sequence. It includes a moiety exhibiting a specific polymerase activity of the present invention. More preferably, the present invention is at least 75%, preferably 85%, more preferably 90%, most preferably 95% or more, 96%, 97%, relative to the site exhibiting the polymerase activity of the present invention, It includes a moiety exhibiting polymerase activity having 98% or 99% identity, and in particular a moiety exhibiting a specific polymerase activity of the present invention disclosed herein and defined by its amino acid sequence or a portion thereof.

Unless explicitly stated otherwise, the terms nucleic acid molecule and nucleic acid are used interchangeably in the context of this application.

The terms "nucleic acid" and "nucleic acid" preferably used herein refer to polynucleotides or oligonucleotides such as deoxyribonucleic acid (abbreviation. DNA) and ribonucleic acid (abbreviation. RNA). In addition, the term “nucleic acid” includes a number of nucleic acids. The terms “nucleic acid” and “nucleic acid” should be understood to include equivalents, variants and analogs of DNA or RNA, single and double-stranded polynucleotides or oligonucleotides made from nucleotide analogs. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. Ribonucleotides include adenosine, cytidine, guanosine and uridine. References to nucleic acid molecules such as "polynucleotide" are used in a broad sense to mean two or more nucleotides or nucleotide analogues linked by covalent bonds, including single-stranded or double-stranded molecules. The term “oligonucleotide” is used herein to mean an oligonucleotide comprising about a hundred nucleotides, as defined herein, but two or more nucleotides or nucleotide analogues linked by covalent bonds.

A nucleic acid is characterized in that all contiguous nucleotides forming the nucleic acid are linked or linked to each other by one or more covalent bonds. Specifically, each of these nucleotides is bound or linked to two other nucleotides, and preferably forms a length of consecutive nucleotides by a phosphodiester bond or other bond. However, in this arrangement, the two terminal nucleotides, i.e., preferably the nucleotides at the 5'end and the 3'end, are linked to each other to a single nucleotide under the condition that the arrangement is a linear arrangement rather than a circular shape. It is a linear molecule rather than a molecule.

In another embodiment of the invention, the nucleic acid comprises at least two groups of contiguous nucleotides, whereby each nucleotide in each group of contiguous nucleotides is linked or linked to two other nucleotides, preferably Phosphodiester linkages or other linkages form the length of contiguous nucleotides. However, in this arrangement, the two terminal nucleotides, ie, preferably at the 5'end and the 3'end, are linked to each other only on a single nucleotide. However, in an embodiment, the two groups of the contiguous nucleotide are bonded to each other or not linked to each other through a covalent bond that binds one nucleotide of one group and the other of the group, or to another group through a covalent bond. It is a covalent bond formed between a sugar moiety of one of the two nucleotides and a phosphor moiety of the other of the two nucleotides. However, in an alternative embodiment, the two groups of contiguous nucleotides are bonded or linked to each other through a covalent bond that binds one nucleotide of one group and the other of one group, or different through a covalent bond. It is bonded to or linked to a group, and is preferably a covalent bond formed between a sugar moiety of one of the two nucleotides and a phosphor moiety of the other of the two nucleotides. Preferably, at least two groups of contiguous nucleotides are not linked through any covalent bond. In another preferred embodiment, at least two groups are bonded through a covalent bond different from the phosphodiester bond.

In addition, the term nucleic acid preferably includes D-nucleic acid or L-nucleic acid. Preferably, the nucleic acid is an L-nucleic acid. In addition, it is possible that one or several portions of the nucleic acid exist as D-nucleic acid, and at least one or several portions of the nucleic acid are L-nucleic acid. The term “part” of a nucleic acid means as little as one nucleotide. Nucleic acid in this specification generally refers to L- and D-nucleic acids, respectively. Thus, in a preferred embodiment, the nucleic acids of the invention consist of L-nucleotides and contain at least one D-nucleotide. Preferably, the D-nucleotide is attached to the ends of any length and also to the ends of any nucleic acid.

L-nucleic acid as used herein is a nucleic acid composed of L-nucleotides, preferably completely composed of L-nucleotides.

D-nucleic acid as used herein is a nucleic acid consisting of D-nucleotides, preferably completely consisting of L-nucleotides.

Also, unless stated otherwise, any nucleotide sequence is described herein in the 5'→ 3'direction.

Nucleic acid regardless of whether the nucleic acid is composed of D-nucleotides, L-nucleotides or combinations thereof, e.g., random combinations or a predetermined sequence of length consisting of at least one L-nucleotide and at least one D-nucleotide The molecule may be composed of deoxyribonucleotide(s), ribonucleotide(s), or a combination thereof.

Regardless of whether the nucleic acid is D-nucleic acid, L-nucleic acid, mixtures thereof, DNA or RNA, or each and any combination thereof, the term nucleic acid preferably used herein is a single-stranded nucleic acid and a double- The nucleic acid molecule comprising a stranded nucleic acid, thereby preferably carrying out the method of the invention, is a single-stranded nucleic acid.

The term nucleic acid preferably used herein includes a modified nucleic acid, the modified nucleic acid may be a nucleotide-modified RNA or a nucleotide-modified DNA molecule, whereby the RNA or DNA molecule is a nuclease resistance group (nuclease resistance group). resistant groups), e.g. 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (see Usman & Cedergren, 1992) at each nucleotide. It can be deformed to improve stability by deformation.

The term nucleic acid preferably used herein includes completely closed nucleic acids. Completely closed, i.e., means a circular structure for a nucleic acid, when the nucleotide sequence in which the nucleic acid is to be determined according to the present invention is closed, preferably through a covalent bond, thereby more preferably as disclosed herein Likewise, a covalent bond is created between the 5'and 3'ends of the nucleic acid molecule sequence.

The term nucleic acid preferably used herein includes any nucleic acid molecule containing a non-nucleic acid molecule moiety. Such non-nucleic acid molecular moieties can be selected from the group comprising peptides, oligopeptides, polypeptides, proteins, carbohydrates, and various groups detailed below. In addition, the term nucleic acid e includes conjugates and/or complexes comprising at least one nucleic acid site and at least one additional site that can be used to facilitate delivery of a nucleic acid molecule into a biological system such as a cell. Provided conjugates and complexes can confer therapeutic activity by delivering therapeutic compounds across cell membranes, altering pharmacokinetics and/or modulating the location of the nucleic acids of the invention. Kinds of such conjugates and complexes are preferably suitable for the delivery of molecules and are small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, such as proteins, Peptides, hormones, carbohydrates, polyethylene glycols, or polyamines that cross cell membranes. In general, the transporters described above are designed to be used individually or as part of a multi-component system, with or without a degradable linker. These compounds are expected to enhance the delivery and or localization of nucleic acid molecules to multiple cell types derived from other tissues, with or without serum (see US 5,854,038). The conjugates of the molecules described herein can be attached to the bioactive molecule through a biodegradable linker such as a biodegradable nucleic acid linker molecule.

As will be described in detail below with respect to the nucleic acid sequence to be determined, the non-nucleic acid moiety may be a PEG moiety, i.e. a poly(ethylene glycol) moiety or a HES moiety, i.e. a hydroxyethyl starch moiety. .

The non-nucleic acid moiety and the preferred PEG and/or HES moiety can be attached directly to the nucleic acid molecule or via a linker. It is also within the scope of the present invention that the nucleic acid molecule comprises one or more modifications, preferably one or more PEG and/or HES moieties. In an embodiment individual linker molecules attach one or more PEG moieties or HES moieties to the nucleic acid molecule. The linker itself used in connection with the present invention may be straight or branched. Linkers of this kind are known to those of ordinary skill in the art, and are described in WO 2005/074993 and WO 2003/035665 patent applications.

In a preferred embodiment, the linker is a biodegradable linker. Due to the release of the modification from the nucleic acid molecule, the biodegradable linker can modify the properties of the nucleic acid molecule in the animal body in terms of the residence time, and is preferably a human body. The use of biodegradable linkers can allow better control of the retention time of the nucleic acid molecule. In a preferred embodiment, such a biodegradable linker is described in international patent applications WO 2006/052790, WO 2008/034122, 2005/099768 and WO 2004/092191, but is not limited thereto, whereby international patent applications WO 2004/092191 and In WO 2005/099768 the linker is part of a polymeric oligonucleotide prodrug and, as described herein, consists of one or two modifications between the nucleic acid molecule and the biodegradable linker.

“Nucleotide” as preferably used herein includes, but is not limited to, naturally occurring DNA nucleoside mono-di, and triphosphate: deoxyadenosine mono-, di- and triphosphate; Deoxyguanosine mono-, di- and triphosphate; Deoxythymidine mono-, di- and triphosphate; Deoxycytidine and mono-, di- and triphosphates. (referred to as dA, dG, dT and dC or A, G, C and T respectively). In addition, the term nucleotide includes mono-, di-, and triphosphates of naturally occurring RNA nucleosides: adenosine mono-, di- and triphosphate; Guanine mono-, di- and triphosphate; Uridine mono-, di- and triphosphate; And cytidine and mono-, di- and triphosphates (respectively referred to herein as A, G, C and U) refer to a base-sugar-phosphate combination, the combination being a monomer unit of a nucleic acid molecule, i.e., DNA Molecule or RNA molecule. However, that is, the term "nucleotide" refers to a cyclic furanoside-type having a purine or pyrimidine-type base attached to the C-1' sugar position through a -glycosyl C1'-N bond and phosphorylated at the 5'position. It means any compound comprising a sugar (pD/L-ribose of RNA and PD/L-2'-deoxyribose of DNA). Nucleotides are mass-modified nucleotides, inter alia, a denatured base (e.g., 5-methyl cytosine) and a denatured sugar group (e.g., 2'-0-methyl ribosyl, 2'-0-methoxyethyl ribosyl, 2'- It may be natural or synthetic, including nucleotides with nucleosides modified with fluoro ribosyl, 2′-amino ribosyl, etc.).

The term "nucleobase" refers to the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), as well as non-naturally occurring nucleobases xanthine, Diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanosytosine, N6,N6 ethano-2,6-diaminopurine, 5-methyl Cytosine, 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazoropyridine, isocytosine, iso Guanine, inosine, and non-naturally occurring nucleobases are described in US Pat. No. 5,432,272 and Freier & Altmann (1997). Thus, the term "nucleic acid base" includes the known purine and pyrimidine heterocycles, as well as heterocyclic analogs and tautomers thereof.

It is within the scope of the present invention that single-stranded nucleic acids are capable of forming unique and stable three-dimensional structures and specifically bind to target molecules such as antibodies. Nucleic acid molecules composed of D-nucleotides are called aptamers. Aptamers can be identified for various target molecules such as small molecules, proteins, nucleic acids, cells, tissues and organisms, and can inhibit the in vitro and/or in vivo functions of specific target molecules. Aptamers are generally identified by a target-direct screening process called in vitro screening or SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (Bock et al, 1992; Ellington & Szostak, 1990; Tuerk & Gold, 1990). Non-denaturing aptamers are quickly removed from the bloodstream with half-life of minutes to hours, mainly due to nuclease degradation and removal from the body by the kidneys as a result of the inherent low molecular weight of the aptamer. Thus, in order to use therapeutic aptamers, they must be modified in the 2'position of the sugar (eg ribose) backbone (Burmester et al., 2006).

The ubiquitous nuclease that accounts for the instability of aptamers is composed of chiral building blocks, i.e., L-amino acids. Thus, the structure of nucleases is chiral in nature, which leads to the recognition of stereospecific substrates. Thus, these enzymes only accept substrate molecules in an appropriate chiral arrangement. Since aptamers and naturally occurring nucleic acid molecules are composed of D-nucleotides, L-oligonucleotides must escape from enzymatic recognition and subsequent degradation. On the same principle, in this case, unfortunately, nature does not develop enzymatic activities that amplify enantiolic nucleic acids. Therefore, L-nucleic acid aptamer cannot be obtained directly using the SELEX process. Although it is a three-dimensional principle, it can be seen a detour to finally reach the desired functional L-nucleic acid aptamer.

When the in vitro selected (D-) aptamer binds to its natural target, the structural mirror image of this aptamer binds to the mirror image of the natural target with the same characteristics. Here, both interacting partners have the same (unnatural) chirality. The homochirality and most biochemical compounds of organisms, enantio-RNA ligands, may of course be limited to practical use. On the other hand, if the SELEX process is performed on a (non-natural) mirror image target, an aptamer recognizing such a (non-natural) target may be obtained. The aptamer-desired L-aptamer-recognition of the corresponding mirror image in turn recognizes a natural target. The first enantioselection process for the generation of biostable nucleic acid molecules was first published in 1996 (Klussmann et al., 1996; Norte et al., 1996), and functional enantiolic nucleic acids exhibiting biological stability as well as high affinity and specificity for target molecules It led to the production of molecular ligands. It is within the scope of the present invention that a single-stranded nucleic acid molecule is a ligand-binding L-nucleic acid called'Spiegelmer' (German word'Spiegel', mirror) (see The Aptamer Handbook'; eds. Klussmann, 2006).

Among them, the nucleic acid of the present invention may contain modifications, and preferably, the nucleic acid of the present invention is modified to be detected. These modifications are preferably selected from the group consisting of radioactive, enzymatic and fluorescent labels. In addition, the modification is selected from D-nucleotides that can be modified by modifications selected from the group consisting of self-radioactive, enzymatic and fluorescent labels.

The chemical properties of the various sequence numbers, nucleic acids, peptides, oligopeptides and proteins used herein, their actual sequences and internal reference numbers are summarized in the table below.

It will be understood that the above are representations of the molecule, as used in connection with the present invention. As shown in the above table, the attached sequence listing reflects only simple amino acid or nucleotide sequences thereof, and does not reflect any additional characteristics of the molecule.

The invention is further described with the use of figures, examples and sequence listings from which additional features, embodiments and advantages may be selected.
1A shows a composition of a 1-gap D-DNA template for testing the activity of L-polymerase X;
1B shows a composition of a 6-gap D-DNA template for testing the activity of L-polymerase X;
Figures 2a-b show the analysis of the synthetic D-polypeptide product Ac-MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSL-OGp(1) by UPLC (A) and mass spectrometry (B);
3A-B show the analysis of the synthetic D-polypeptide product H-RREEKLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKA-SMe(2) by UPLC (A) and mass spectrometry (B);
Figure 4a-b shows the analysis of synthetic D-polypeptide product H-CGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTGPV SYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELI KELGFTYRIPK by UPLC (A) and mass spectrometry (B);
5A-B show analysis of the synthetic D-polypeptide product Ac-MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKA-SMe (4) by UPLC (A) and mass spectrometry (B);
Figure 6a-b is a SDS-PAGE (A) and mass spectrometry (mass spectrometry) natural and chemical connection of the (B) represents an analysis of the product D- polypeptide Ac-MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKACGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELIKELGFTYRIPKKRL-OH (5);
7 shows a composition of a 1-gap L-DNA template for testing the activity of D-polymerase X;
Figure 8 shows the gel electrophoresis of the L-DNA extension activity assay of D-polymerase X on 1-gap substrate;
9A shows a composition of a 6-gap L-DNA template for testing the activity of D-polymerase X;
Figure 9b shows gel electrophoresis of the L-DNA extension activity assay of D-polymerase X on 6-gap substrate;
10A shows a primer-template complex D-DNA substrate for assaying the activity of L-polymerase X;
Figure 10b shows the gel electrophoresis of the D-DNA extension activity assay of L-polymerase X carried out at a constant temperature;
Figure 10c shows the gel electrophoresis of the D-DNA extension activity assay of L-polymerase X conducted using temperature cycling;
Fig. 11 ab shows the analysis of synthetic all-L-polymerase dpo4 variant A155C by SDS-PAGE (A) and LC-ESI mass spectrometry (B);
12A shows gel electrophoresis of D-DNA PCR activity assays of L-polymerase dpo4 variants A155C, V203C, C31S and A155C/V203C;
12B shows gel electrophoresis of a D-DNA PCR activity assay of recombinant and synthetic L-polymerase dpo4;
13 shows the analysis of the synthetic D-polypeptide product H-RTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-NH2(1) by mass spectrometry;
Figure 14 shows the analysis of synthetic D-polypeptide product Boc-VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRG-OH (2) by mass spectrometry;
Figure 15 shows the analysis of the fragment condensation D- polypeptide product H- VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-NH ₂ (3) due to the mass spectrometer;
Figures 16a-b show the analysis of synthetic D-polypeptide product Z-CDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKL-benzyl-thioester (4) by RP-HPLC (A) and mass spectrometry (B);
Figures 17a-b show the analysis of the synthetic D-polypeptide product H-RKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIA-SMe (7) by UPLC (A) and mass spectrometry (B);
Figure 18 ab shows the analysis of the synthetic D-polypeptide product Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPM-OGp (6) by UPLC (A) and mass spectrometry (B);
Figure 19 ab shows the analysis of the Clostripine-mediated D-polypeptide ligation product Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQKAGIPIVEAKKILPNAVYLPMRKEVYQQVKAGIPIVEAKKILPNAVYLPMRKEVYQQVKAGIPIVEAKKILPNAVYLPMRKEVYQNKQVSSRIMNLKLKDK
Figure 20 ab shows the natural chemical linkage product of the all-L-polymerase dpo4 fragment 155-352 (V203C) by SDS-PAGE (A) and LC-ESI mass spectrometry (B). Indicates analysis.

Example

In this example, the following abbreviations are used.

ACN: acetonitrile (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

DCM: dichloromethane (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

DIPEA: N,N-diisipropylamine (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

EDT: EDT (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

Fmoc: 9-Fluorenyl-methoxycarbonyl-

HATU: (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (CreoSalus, Louisville KY, USA)

HFIP: 1,1,1,3,3,3-hexafluorophosphate (1,1,1,3,3,3-hexafluoro phosphate) (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

HPLC: High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography)

MeIm: methyl imidazole (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

MeOH: methanol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

MSNT: 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole) ( Merck KGaA, Darmstadt, Germany)

NMP: N-methyl-pyrrolidone (Iris Biotech GmbH, Marktredwitz, Deutschland)

PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate ((Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphat) (Merck KGaA, Darmstadt, Germany)

SDS: Sodium dodecyl sulfate (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

TBTU: O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (O-(Benzotriazol-1-yl)-N,N,N', N'-tetramethyluroniumtetrafluoroborat) (Merck KGaA, Darmstadt, Germany)

tBu: tert.-butyl-(tert.-Butyl-)

TFA: trifluoroacetic acid (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

TFE: 1,1,1-trifluoroethanol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

THF: THF (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

TIS: TIS (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

TLC: Thin layer chromatography

Tris: Tris (hydroxymethyl) aminomethane (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland)

UPLC: Ultra-performance liquid chromatography

Example 1. Recombinant expression and purification of wild-type and polymerase X variants

Polymerase X derived from African swine fever virus (ASFV) was described and specified by Oliveros et al. in 1997. The wild-type gene of polymerase X has an open reading frame (abbreviation ORF) of only 525 base pairs including a start codon and a stop codon (Oliveros et al, 1997). . The encoded protein is only 174 amino acids long. This example describes how polymerase X as well as its variants are expressed in E. coli and purified using His ₆ -Tag.

1.1 Expression structure

Since the codon usage of ASFV is different from E. coli, an E. coli-codon-optimized synthetic gene for polymerase X was purchased from GeneArt AG (Regensburg, Germany). This synthetic gene sequence was provided in the pCR4-Blunt-TOPO vector (originator company: Invitrogen, Karlsruhe, Germany). The codon-optimized open reading frame containing a start codon and two stop codons had the following sequence:

.

To obtain the expression structure for polymerase X, also referred to as all-L polymerase X, the gene of polymerase X was cut from pCR4-Blunt-TOPO using BamHI and PstI, and pRSET- It was subcloned into A vector (Invitrogen, Karlsruhe, Germany). Subcloning added His ₆ -Tag at the N-terminus and brought the gene under the control of the T7 promoter. The structure was named pMJ14, and was used for the expression of all-L polymerase X (all-L polymerase X) in E. coli. Protein polymerase X expressed from pMJ14 had the following 210 amino acid sequence:

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSMLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKVCGERKCVLFIEWEKKTYQLDLFTALAEEKPLRAIFHKFTKNYKLIKENIFALKFTKILIKE

The first 36 amino acids exhibited His _6- Tags, including several spacer amino acids and some other sequence tags (T7 Gene 10 leader, Anti-Express Epitope). The final 174 amino acid portion was identical to the polymerase X protein sequence found in ASFV.

The expression structure for a variant of all-L polymerase X was made according to the manufacturer's protocol using a commercially available QuikChange kit (Stratagene GmbH, Waldbronn, Germany). Plasmid pMJ14 was used as a template. Oligonucleotides required for QuikChange were synthesized at NOXXON facility (QC10_up, QC10_low) or purchased from Purime (Grebenstein, Germany) (QC26_up, QC26_low, QC27_up, QC27_low, QC31_up, QC31_low). The following variant expression construct was created and used for the expression of a variant of all-L polymerase X in E. coli:

Variant Expression structure Oligonucleotide structure used for the QuikChange mutagenesis procedure I124G pMJ130 QC10_up
(5'- TCCGGTGAGCTATCTGGGTCGTATTCGTGCGGCG-3')
QC10_low
(5'- CGCCGCACGAATACGACCCAGATAGCTCACCGGA-3') V80G pMJ356 QC26_up
(5'- TGAGCTTTAGCGTGAAAGGGTGCGGCGAACG-3')
QC26_low
(5'- CGTTCGCCGCACCCTTTCACGCTAAAGCTCA-3') V80A pMJ357 QC27_up
(5'- TGAGCTTTAGCGTGAAAGCGTGCGGCGAACG-3')
QC27_low
(5'- CGTTCGCCGCACGCTTTCACGCTAAAGCTCA-3') C86S pMJ412 QC31_up
(5'- TGAAAGTGTGCGGCGAACGTAAAAGCGTGCTGTTTA-3')
QC31_low
(5'- TAAACAGCACGCTTTTACGTTCGCCGCACACTTTCA-3')

1.2 Protein expression in E. coli

All-L polymerase X (all-L polymerase X) was expressed in E. coli using the expression structure pMJ14. Variants of all-L polymerase X were expressed from pMJ130, pMJ356, pMJ357 or pMJ412. For expression, a water-soluble E. coli strain'BL-21 (DE3) pLysS' (Novagen/VWR, Dresden, Germany) was transformed with an appropriate expression structure, and the antibiotic Ampicillin was maintained. The culture was grown in 2YT medium at 37° C. until the optical density reached about 0.6 at 600 nm. Protein expression was induced by adding isopropyl beta-D-1-thiogalactopyranoside (abbreviation IPTG) to a final concentration of 0.4 mM. Expression was carried out at 30° C. for 4 hours. Cells were recovered by centrifugation and stored at -80°C or processed immediately.

1.3 Protein purification

Resuspend fresh or frozen E. coli cells on ice in'lyse and bind buffer' (50 mM Na-Phosphate, pH 7.5, 500 mM NaCl, 40 mM imidazole) (resuspended), and lysed using a'French Press' (G. Heinemann, Schwaeisch Gmuend, Germany) cell disruptor. Purification was performed at 4° C. using a'Ni-NTA Superflow' material (Qiagen, Hilden, Germany). Step elution was performed using an elution buffer (50 mM sodium phosphate (Na-Phosphate), pH 7.5, 500 mM NaCl, 200 mM imidazole). Fractions were analyzed using SDS-PAGE (Invitrogen, Karlsruhe, Germany), pooled, and if necessary, anion in the'AKTA purifier' system using'Q Sepharose fast flow' material (GE healthcare, Freiburg, Germany). -Further purification was performed through anion-ion-exchange chromatography. Protein identity was confirmed using MALDI mass spectometry, and appropriate fractions were pooled, concentrated, and re-buffered. The purified protein was stored at -20 °C in a buffer consisting of 25 mM sodium phosphate (Na-Phosphate), pH 7.5, 250 mM NaCl, and 50% glycerol. Protein concentration was measured by BCA-protein analysis (Pierce/Perbio Science, Bonn, Germany) using bovine serum albumin (abbreviated BSA) standards.

Example 2. Confirmation of the activity of polymerase X and variants of polymerase X

Activity assays for all-L polymerase X and variants of all-L polymerase X (see Example 1) were performed using different types of substrate complexes formed by oligonucleotides. Performed, where the substrate and oligonucleotide are composed of D-DNA-nucleotides.

2.1 Activity assay for substrates with 1-nucleotide gaps

List of oligonucleotides for 1-gap substrates: designation Nucleotide length Sequence (5'→3') SP-1 15 GATCACAGTGAGTAC D(g1)P 17 Phosphate-GTAAAACGACGGCCAGT MJ_1_140_DD 33 ACTGGCCGTCGTTTTACAGTACTCACTGTGATC MJ_1_141_DD 33 ACTGGCCGTCGTTTTACCGTACTCACTGTGATC MJ_1_142_DD 33 ACTGGCCGTCGTTTTACGGTACTCACTGTGATC SP1c+18(g1) 33 ACTGGCCGTCGTTTTACTGTACTCACTGTGATC

The substrate complex was hybridized to the template strand at each of the 5'and 3'ends, with two different DNA nucleotides consisting of 15 and 17 nucleotides, respectively, of a DNA oligonucleotide consisting of 33 nucleotides. It was prepared by annealing of the template strand (also called the lower strand), and a gap of one nucleotide was created in the upper strand. The composite contained A, C, G, or T in the template position in the gap. Prior to annealing, by standard kinase reaction using gamma- ³² P-adenosine-triphosphate (ATP) and T4 polynucleotide kinase, the oligonucleotide SP-1 consisting of 15 nucleotides ³² It was radiolabeled at its 5'-end with P. Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. Gamma- ³² P-ATP, which was not used for labeling, was purified and removed with NAP-column (GE healthcare).

In the activity assay, all-L polymerase X and its variants were bound with a D-configurated 1-gap substrate complex (see Fig. 1A). As a negative control, each substrate was also incubated without all-L polymerase X, its variants and D-dNTP's (D-desoxy-nucleotide-triphosphates). Depending on the template base in the 1-gap complex, only the corresponding D-dNTP was added during the analysis. A typical 6 μl assay is a 50 nM substrate complex, 1.7 ng/μl L-all-L polymerase X or a variant thereof, 8 μM of one D-dNTP and buffer (50 mM Tris -HCl, 10 mM MgCl ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). D-dNTP's were purchased from Rovalab (Teltow, Germany). The incubation time was 30 minutes at 37°C. The entire assay volume was mixed with sample buffer/dye, loaded onto a denatured sequencing gel, and separated for 4 hours. The gel was exposed to Kodak K overnight at -80° C. and read using the BioRad Fx phosphoimager system.

All-L polymerase X and its variants I124G, V80A and V80G were active under these conditions and filled the 1 nucleotide gap between the two upper strand DNA oligonucleotides.

2.2 Activity assay for substrates with 6-nucleotide gaps

List of oligonucleotides for 6-gap substrates: designation Nucleotide length Sequence (5'→3') SP-1 15 GATCACAGTGAGTAC D(g6)P 12 Phosphate-ACGACGGCCAGT SP1c+18(g6) 33 ACTGGCCGTCGTTCTATTGTACTCACTGTGATC

The substrate complex was hybridized to the template strand at each of the 5'and 3'ends, with two different DNA nucleotides consisting of 15 and 12 nucleotides, respectively, of a DNA oligonucleotide consisting of 33 nucleotides. It was prepared by annealing of the template strand (also called the lower strand), and a six nucleotide gap was created in the upper strand. Prior to annealing, by standard kinase reaction using gamma- ³² P-adenosine-triphosphate (ATP) and T4 polynucleotide kinase, the oligonucleotide SP-1 consisting of 15 nucleotides ³² It was radiolabeled at its 5'-end with P. Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. Gamma- ³² P-ATP, which was not used for labeling, was purified and removed with a NAP-column (GE healthcare, Freiburg, Germany).

In the activity assay, all-L polymerase X and variants thereof were combined with a D-configurated 6-gap substrate complex (see Fig. 1B). As a negative control, each substrate was also incubated without all-L polymerase X, its variants and D-dNTP's (D-desoxy-nucleotide-triphosphates). A typical 6 μl assay is a 50 nM substrate complex, up to 1.3 ng/μl all-L polymerase X (all-L polymerase X) or a variant thereof, 8 μM of each D-dNTP and buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). D-dNTP's were purchased from Rovalab (Teltow, Germany). Typical incubation time was 30 minutes at 37°C. The entire assay volume was mixed with sample buffer/dye, loaded onto a denatured sequencing gel, and separated for 4 hours. The gel was exposed to Kodak K overnight at -80° C. and read using the BioRad Fx phosphoimager system.

All-L polymerase X and its variant (C86S) were active under these conditions and filled the 6 nucleotide gap between the two upper strand DNA oligonucleotides.

Example 3. Synthesis of a variant of the polymerase Pol X consisting of D-amino acid

In this example, the synthesis of all-D polymerase X variant V80A is described. The amino acid sequence of the all-D polymerase X variant V80A is Ac-MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKACGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTGPVSYKLIQRATTFNYKKLIQRIKTL.

All amino acids used are protected according to the Solid-phase peptide synthesis Fmoc/tBu-strategy requirements ( Eric Atherton et al., 1981). All amino acids used are D-amino acids (Bachem, Bubendorf, Switzerland).

3.1 HO-Gp(Boc) ₂ Synthesis of

tert - butyloxycarbonyl - such as a gavel no phenol obtain a protected 4- (tert -butyloxycarbonyl-protected 4- guanidinophenol) Sekizaki (. Sekizaki et al, 1996) and were synthesized in a similar manner. According to this, 40 mmole N,N' -bis( tert - butyloxycarbonyl)-S-methylisothiourea ( N,N'- Bis-( tert- butyloxycarbonyl)-S-methylisothiourea) (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) and 60 mmole 4-aminophenol (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland) were dissolved in 250 ml THF in a 500 ml round bottom flask. Then, the solution was flushed with argon for 10 minutes, and stirred for 120 hours while sealed with a calcium chloride (CaCl ₂ ) tube.

After evaporation of the solvent, the residue was precipitated with ice-cold methanol. The precipitate was dried under vacuum over P ₄ O ₁₀ . Finally, the product was purified using flash chromatography using DCM. The product containing fractions was collected and the solvent was evaporated under reduced pressure. For the analysis, TLC, reverse phase HPLC, mass spectrometry and NMR were used. The experimentally determined amount corresponds to a calculated amount of 351 Da.

3.2 H-d-Leu-OGp(Boc) ₂ Synthesis of

1 mmole Zd-Leu-OH (Bachem, Bubendorf, Switzerland), 0.9 eq. TBTU and 0.9 eq. HO-Gp(Boc) ₂ was dissolved in 10 ml DMF. 2 eq. After adding DIPEA, the solution was stirred for 2 hours. After evaporation of the solution, the raw product was purified by flash chromatography using DCM. Pure fractions of Zd-Leu-OGp(Boc) ₂ were collected and the solvent was evaporated.

Zd-Leu-OGp(Boc) ₂ was dissolved in 10 ml methanol (MeOH) and flushed with argon. Hydrolytic cleavage of the N-terminal Z-group was carried out for 2 hours by adding a Pd/C catalyst and H ₂ . After Hd-Leu-OGp(Boc) ₂ was filtered, methanol (MeOH) was evaporated under reduced pressure. Analysis was carried out using reverse phase HPLC and mass spectrometry. The correct mass of 465 Da for the product was found, which corresponds to the calculated mass.

3.3 Synthesis of all-D-peptide AcMLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNI VAVGSL-OGp(1)

0.10 mmole TentaGel-R-Trityl resin (Rapp Polymere, Tuebingen, Deutschland) was added to Fmoc-d-Ser(tBu)-OH (Bachem, Bubendorf, Switzerland) as described by Barlos et al., 1989. Loaded with. Therefore, 0.10 mmol resin was incubated twice with 0.6 mmole thionylchloride for 30 minutes, followed by washing with DCM. Then, the resin was incubated with 0.6 mmole Fmoc-d-Ser(tBu)-OH, 2.4 mmol DIPEA in 6 ml DCM for 90 minutes. Then, the resin was blocked three times for 10 minutes using a 10% MeOH (v/v), 10% DIPEA (v/v) solution in DCM, and washed with DCM. Automatic synthesis was performed using ABI 433 (Applied Biosystems, Foster City, USA) having the FASTmoc protocol. 10 eq. Amino acids are 9 eq. in NMP. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed 3 times for 7 minutes with 20% (v/v) piperidine in NMP (Sigma-Aldrich Chemie GmbH, Schnelldorf, Deutschland). I did.

Cleavage of fully protected peptide acid was achieved by incubating peptidyl resin for 2 hours in 30% (v/v) HFIP in 10 ml of DCM. After filtering the peptide, the solvent was evaporated and the residue was precipitated using ice-cold diethyl ether. The precipitated peptide was separated and dried.

0.01 mmole of fully protected peptide, 4 eq. PyBOP and 5 eq. Hd-Leu-OGp(Boc) ₂ was dissolved in 6 ml NMP. 10 eq. After DIPEA was added, the mixture was stirred for 4 hours. Then, the solvent was evaporated under reduced pressure and the residue was precipitated with ice-cold diethyl ether. The precipitated peptide ester was dried and then the protection groups were cleaved for 2 hours using 2.5% EDT, 2.5% water, and 2.5% TIS. After evaporation of the TFA, the peptide was precipitated with ice cold diethyl ether. Reverse phase HPLC purification of the peptide ester was performed using an ACN/water gradient on a C18 column (Phenomenex, Aschaffenburg, Germany). Fractions containing the product were collected and freeze-dried.

The final product was characterized by HPLC/UPLC (Figure 2A) and mass spectrometry (Figure 2B). The measured mass of 4654.7 Da for the product corresponds to the theoretical mass of 4652.7 Da.

3.4 Synthesis of all-D-peptide H-RREEKLNDVDLLIIVPEKKLLKHVLPNIRIKGLSF SVKA-SMe(2)

0.10 mmole TentaGel-R-NH ₂ resin (Rapp Polymere, Tuebingen, Deutschland) was added to 5 eq. in 6 ml NMP. Amino acids, eq. 4.9 eq. HATU and 10 eq. It was loaded for 45 minutes with Fmoc-d-Ala-OH using DIPEA. The resin was then washed with THF. Conversion to Fmoc-d-Ala-ψ[CS-NH]-R-TentaGel was carried out at 80° C. for 2 hours at 4 eq. This was done by incubation with Lawesson's reagent. Then, the resin was washed with NMP. The resin thus prepared was then used for automatic peptide synthesis as described above (see Example 1.3).

Then, the corresponding thioester was prepared by incubating with methyl iodide in DMF overnight according to Sharma et al. (Sharma et al., 2011). After filtering the resin, the peptide thioester containing the solvent was evaporated and the residue was precipitated using ice cold diethyl ether. Cleavage of side chain protection groups was performed for two hours with 2.5% EDT, 2.5% water and 2.5% TIS in TFA. After evaporation of the TFA, the peptide was precipitated with ice cold diethyl ether. Reverse phase HPLC purification of the peptide thioester was performed on a C18 column (Phenomenex, Aschaffenburg, Germany) using an ACN/water gradient. Fractions containing the product were collected and freeze-dried.

The final product was characterized by HPLC/UPLC (Figure 3A) and mass spectrometry (Figure 3B). The measured mass for the product (4683.8 Da) corresponds to the theoretical value of 4681.7 Da.

3.5 Synthesis of all-D-peptide H-CGERKCVLFIEWEKKTYQLDLFTALAEEKPYA IFHFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELI KELGFTYRIPKKRL-OH(3)

0.10 mmole TentaGel-R-PHB resin (Rapp Polymere, Tuebingen, Deutschland) was added to 6 eq. in DCM. amino acid, 6 eq. Loaded with Fmoc-d-Leu-OH using MSNT and 4.5 eq MeIm. Automatic synthesis was performed using ABI 433 with FASTmoc protocol. 10 eq. Amino acids are 9 eq. in NMP. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP. A double coupling step was performed after 42 amino acids. Cleavage of the N -terminal Fmoc-protected peptide was performed using 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. Following evaporation of TFA, the peptide was precipitated with ice cold diethyl ether. Reverse phase HPLC purification of the crude N -terminal Fmoc-protected peptide was performed on a C18 column (Phenomenex, Aschaffenburg, Germany) using an ACN/water gradient. The product containing the fractions was collected and lyophilized. The Fmoc-protected peptide was then dissolved in 20% piperidine in DMF and stirred for cleavage of the N-terminal Fmoc-rrl. After 20 minutes, the solvent was evaporated and the residue was precipitated using ice cold diethyl ether. The precipitated crude peptide was then purified by reverse phase HPLC using a C18 column (Phenomenex, Aschaffenburg, Germany) using an ACN/water gradient. The product containing the fractions was collected and lyophilized.

The final product was characterized by HPLC/UPLC (Figure 4A) and mass spectrometry (Figure 4B). The measured mass for the product (11184.3 Da) corresponds to the theoretical mass of 11178.2 Da.

3.6 Synthesis of all-D-peptide Ac-MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIeKGLSVKA-SM by protease-catalyzed ligation of peptide 1 with peptide 2

Peptide 1 was dissolved at 0.2 mM and peptide 2 at 0.6 mM in sodium phosphate buffer (100 mM, pH 8.5, with 100 mM NaCl) containing 2% Triton X100 (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany). After adding 20 µl Clostripain (Endoprotease Arg-C, Worthington Biochemical Corporation, Lakewood, NJ, USA), the reaction mixture was shaken at 37° C. overnight. The precipitated peptide was centrifuged, dissolved in H ₂ 0/ACN/formic acid 60/40/0.5, and RP-18-column with a gradient of ACN in water of 30% to 60% within 30 minutes (Phenomenex, Aschaffenburg, Germany). The product containing the fractions was collected and lyophilized.

The final peptide was analyzed by reverse phase UPLC (Figure 5A) and ESI-mass spectrometer (Figure 5B). Theoretical molecular weight (M _theor = 9199.3 Da) corresponds to the observed molecular weight (M _obs = 9199.4 Da).

3.7 Synthesis of all-D polymerase X (all-D polymerase X) variants by natural chemical ligation of peptide 4 with peptide 3

Peptides 3 and 4 were all 6 M guanidine hydrochloric acid (GuanidinHCl) (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany), 200 mM mercaptophenyl-acetic acid (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) and 5 mm Dissolved to 0.2 M in TRIS-buffer (pH 8.6) containing Tris(2-carboxyethyl)phosphine hydrochloride (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) Made it. The reaction mixture was shaken at room temperature for 72 hours. The mixture was then purified by reverse phase HPLC using RP-8-columns (Phenomenex, Aschaffenburg, Germany) with a gradient of ACN in 30% to 60% water within 30 minutes. Fractions containing a ligation product were collected and dried. The dry powder was dissolved in water and a SEC3000-column (sodium-buffer phosphate) (Sigma Aldrich Chemie GmbH, Schnelldorf, Germany) (50 mM, pH 6.8, 0.5% SDS) as an eluent ( Phenomenex, Aschaffenburg, Germany) was purified by size exclusion chromatography. The product containing the fractions was collected and lyophilized.

The final product was analyzed by SDS-PAGE (Figure 6A) and ESI-mass spectrometer (Figure 6B). A clear band was found in lane 7 between 14.4 kDa and 21.5 kDa indicating pure full length polymerase. Theoretical molecular weight (M _theor = 20342 Da) corresponds to a molecular weight (M _obs = 20361 Da) observed seen by the ESI-MS.

Example 4. Confirmation of activity of synthetic polymerase X variant composed of D-amino acid

The dried all-D polymerase X variant V80A according to Example 3 was dissolved in 6 M guanidinium hydrochloride and commercially available having a molecular weight of 3,500 cut-off. In one medical dialysis device (Pierce/PerBio, Bonn, Germany), it was refolded at 4° C. by stepwise dialysis. The final buffer is 50 mM sodium phosphate, 500 mM sodium chloride, pH 7.5. The protein concentration was determined by a standard series of known proteins according to SYPRO-RED staining (Invitrogen, Karlsruhe, Germany) and pre-cast gels using a densiometric band on a BioRad Fx scanner instrument (Invitrogen, Karlsruhe. , Germany) on SDS-PAGE.

Activity assays for the all-D polymerase X variant V80A were performed with two different substrate types:

4.1 Activity assay for substrates with 1-nucleotide gap

The substrate was prepared by annealing a 33-mer lower strand DNA oligonucleotide having a 17-mer upper strand DNA, and the gap of one nucleotide in the upper strand Was made. Prior to annealing, 17-mer upper strand oligonucleotide MJ_1_58_MD by standard kinase reaction using gamma- ³² P-adenosine-triphosphate (ATP) and T4 polynucleotide kinase. Was radiolabeled at its 5'-end with ³² P. To facilitate the kinase reaction of L-oligonucleotides, two D-configurated guanosine bases were added to the 5'end during oligonucleotide synthesis. Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. Gamma- ³² P-ATP, which was not used for labeling, was purified and removed with a NAP-column (GE healthcare, Freiburg, Germany). The composite included A, C, G or T in the template position within the gap. See FIG. 7 for the setup of the substrate complex.

In the activity assay, synthetic all-D polymerase X (all-D polymerase X) variant V80A was combined with an L-configurated 1-gap substrate complex. As a negative control, each substrate was also incubated without the pre-D polymerase X variant V80A and L-dNTP's (L-desoxy-nucleotide-triphosphates). Depending on the template base in the 1-gap complex, only the corresponding D-dNTP was added during the analysis. A typical 6 μl assay is 50 nM substrate complex, 1.7 ng/μl all-D polymerase X variant V80A, 8 μM of one L-dNTP and buffer (50 mM Tris-HCl, 10 mM MgCl). ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). L-dNTP's is owned by Rasayan, Inc. (Encinitas, CA, USA) was purchased by custom synthesis. The incubation time was 30 minutes at 37°C. The entire assay volume was mixed with sample buffer/dye, loaded onto a denatured sequencing gel, and separated for 4 hours. The gel was exposed to Kodak K overnight at -80° C. and read using the BioRad Fx phosphoimager system.

As shown in FIG. 8, the all-D polymerase X (all-D polymerase X) variant V80A provides an extension product to the L-DNA 1-gap substrate, thus confirming the activity of the synthetic protein. Notably, only the all-D polymerase X (all-D polymerase X) variant V80A combined with the L-substrate and L-dNTP's provides any elongation product. In addition, only samples containing L-dNTP corresponding to their template base obtained an elongation product. It means that A-complex dTTP nucleotides, C-complex dGTP nucleotides, G-complex dCTP nucleotides, and T-complex dATP nucleotides must be present to obtain any elongation product.

4.2 Activity Assay for Substrates with 6-nucleotide Gap

The substrate was prepared by annealing a 33-mer lower strand DNA oligonucleotide having 17-mer and 12-mer upper strand DNA, and six nucleotides in the upper strand. A gap was created. Prior to annealing, 17-mer upper strand oligonucleotide MJ_1_58_MD by standard kinase reaction using gamma- ³² P-adenosine-triphosphate (ATP) and T4 polynucleotide kinase. It was radiolabeled at its 5'-end with ³² P in (L-configuration). To facilitate the kinase reaction of L-oligonucleotides, two D-configurated guanosine bases were added to the 5'end during oligonucleotide synthesis. Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. Gamma- ³² P-ATP, which was not used for labeling, was purified and removed with a NAP-column (GE healthcare, Freiburg, Germany). See Figure 9A for the setup of the substrate complex.

In the activity assay, the synthetic all-D polymerase X (all-D polymerase X) variant V80A was combined with an L-configurated 6-gap substrate complex. As a negative control, the substrate was also cultured without the pre-D polymerase X variant V80A and L-dNTP's (L-desoxy-nucleotide-triphosphates). A typical 6 μl assay is 50 nM substrate complex, up to 1.3 ng/μl of all-D polymerase X variant V80A, 8 μM of each L-dNTP's and buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5). L-dNTP's is owned by Rasayan, Inc. (Encinitas, CA, USA) was purchased by custom synthesis. Typical incubation times are 30 minutes at 37° C., but longer incubations may be used depending on the activity of the batch. The entire assay volume was mixed with sample buffer/dye, loaded onto a denatured sequencing gel, and separated for 4 hours. The gel was exposed to Kodak K overnight at -80° C. and read using the BioRad Fx phosphoimager system.

9B, synthetic all-D polymerase X (all-D polymerase X) variant V80A provides an N+6 extension product to the L-DNA 6-gap substrate, thus confirming the activity of the synthetic protein. However, the synthesis of the N+6 extension product was less evident than the N+1 extension product for the same 6-gap complex. In addition, increased incubation time was required to fill the 6-gap. Notably, only the all-D polymerase X (all-D polymerase X) variant V80A with L-substrate added to L-dNTP's provides any extension product.

Example 5. DNA synthesis by polymerase X and variants of polymerase X

Polymerase X from African swine fever virus (abbreviation ASFV) is described in Oliveros, 1997 as a widely distributed enzyme with a gap repair function. As shown in Example 2, pre-L-polymerase X and variants thereof catalyze the incorporation of only a very small number of nucleotides after each initiation on gapped substrates. Seems to be. Here, the present inventors disclose a method using all-L-polymerase X and variants to synthesize longer DNA and show complete polymerization of the 83-mer strand.

5.1 Primer-template substrate

A primer-template complex was used to test the activity of pre-L-polymerase X and its variants. The same complex was used to test variants V80G and V80A of pre-L-polymerase X.

List of D-oligonucleotides for primer-template complexes without gaps:

designation Nucleotide length Configuration Sequence (5'→3') MJ_1_33_DD 19 D Atto532-GGAGCTCAGACTGGCACGC MJ_1_1_DD 83 D GTGGAACCGACAACTTGTGCTGCGTCCAGCATAAGAAAGGAGCTCCCTCAGAAGAAGCTGCGCAGCGTGCCAGTCTGAGCTCC

The substrate was prepared by annealing a template-stranded DNA oligonucleotide (MJ_1_1_DD) consisting of 83 nucleotides having a DNA oligonucleotide consisting of 19 nucleotides. Oligonucleotides were synthesized in NOXXON. Oligonucleotide MJ_1_33_DD carries the fluorescent dye Atto-532 (AttoTec, Siegen, Germany). Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. The primer-template complex is depicted in Figure 10A.

5.2 Reaction at constant temperature

In the activity assay, all-L polymerase X and variant V80G or V80A of all-L polymerase X were bound to a D-configurated primer-template complex. . A typical 6 μl assay is a 50 nM substrate complex, up to 1.3 ng/μl of all-L polymerase X or variant V80G or V80A of all-L polymerase X, 8 μM of each D-dNTP's and Buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5) was included. D-dNTP's were purchased from Rovalab (Teltow, Germany). Incubation time for Pol-X samples was 30 minutes at 37°C. As a negative control, the substrate was also incubated without any all-L polymerase X or its variants V80G or V80A of all-L polymerase X and D-dNTP's (D-desoxy-nucleotide-triphosphates). Positive control was performed using Taq polymerase (Invitrogen, Karlsruhe, Germany) used at a final concentration of 0.083 U/µl in Taq buffer supplied by the manufacturer. Taq samples were incubated at 60° C. for 30 minutes. The total assay volume was mixed with sample buffer/dye and separated from the denaturing gel. The gel was read using the BioRad Fx phosphoimager system.

Although all-L polymerase X or variant V80G or V80 of all-L polymerase X was active, polymerization of the entire 83-mer template could not be completed. The Taq polymerase positive control showed complete polymerization of the 83-mer template, as shown in Fig. 10B.

5.3 Reaction under thermal cycling conditions

Assuming that after initiation, all-L polymerase X catalyzes only the incorporation of one nucleotide, and then pauses while staying in the DNA substrate, the present inventors believe that all-L polymerase X is from the template. Repeated heat pulses (50° C., 2 minutes) were performed to allow separation and reconnection. Using this repetitive thermal cycling process, the present inventors were able to perform complete polymerization of 83-mer with all-L polymerase X. Reactions and controls were set up as described above for constant temperature, except that the temperature profile of the pre-L polymerase X sample was run as follows:

5 to 25 cycles (30 min at 20 °C / 2 min at 50 °C)

Then as the final step, 30 minutes at 20 ℃

From 15 cycles, it was observed that all-L polymerase X could polymerize the entire 83-mer template strand, as shown in Fig. 10 similar to the positive control.

Example 6. Primer extension using synthetic polymerase X variant consisting of D-amino acid

The method disclosed in this example uses an L-configurated substrate to test a pre-D configured polymerase.

6.1 Primer-template substrate

List of D-oligonucleotides for primer-template complexes without gaps:

designation Nucleotide length Configuration Sequence (5'→3') MJ_1_109_MD 21 first two = D, others L D(GG)-L(GGAGCTCAGACTGGCACGC) MJ_1_105_LD 83 L GTGGAACCGACAACTTGTGCTGCGTCCAGCATAAGAAAGGAGCTCCCTCAGAAGAAGCTGCGCAGCGTGCCAGTCTGAGCTCC

The substrate was prepared by annealing an 83-mer lower strand DNA oligonucleotide having a 19-mer upper strand DNA oligonucleotide. Oligonucleotides were synthesized in the internal facility of NOXXON by L-configuration. Prior to annealing, by standard kinase reaction using gamma- ³² P-adenosine-triphosphate (ATP) and T4 polynucleotide kinase, 21-mer upper strand oligonucleotide MJ_1_109_MD Was radiolabeled at its 5'-end with ³² P. To facilitate the kinase reaction of L-oligonucleotide MJ_1_109_MD, two D-configurated guanosine bases were added to the 5'end during oligonucleotide synthesis. Annealing was performed by heating at 65° C. for 10 minutes at 10 mM Tris-HCl, 5 mM MgCl ₂ , pH 8.0 and cooling slowly. Gamma- ³² P-ATP, which was not used for labeling, was purified and removed with a NAP-column (GE healthcare, Freiburg, Germany).

6.2 Reaction at constant temperature

In the activity assay, variant V80A of the synthetic all-D polymerase X was combined with an L-configurated primer-template complex. A typical 6 μl assay is a 50 nM substrate complex, up to 1.3 ng/μl of all-L polymerase X or variant V80G or V80A of all-L polymerase X, 8 μM of each L-dNTP's and Buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 4% glycerol, 0.1 mg/ml bovine serum albumin (BSA), pH 7.5) was included. L-dNTP's was purchased from Rasayan, Inc. (Encinitas, CA, USA). The incubation time was at least 30 minutes at 37°C. As a negative control, the substrate was also incubated without any polymerase and L-dNTP's (desoxy-nucleotide-triphosphates). The total assay volume was mixed with sample buffer/dye and separated from the denaturing gel. The gel was read using the BioRad Fx phosphoimager system.

Synthetic all-D polymerase X variant V80A was active under this condition-similar to the all-L polymerase X counterpart-a total of 83 The nucleotide template strand could not be polymerized.

6.3 Reaction under thermal cycling conditions

Similar to Example 5, a repeated thermal cycling process was used to allow the overall polymerization of the 83-mer L-substrate with the all-D Polymerase X (all-D Polymerase X) variant V80A. Reaction and control were set up as described above except that the temperature profile was run as follows at constant temperature:

5 to 25 cycles (30 min at 20 °C / 2 min at 50 °C)

Then as the final step, 30 minutes at 20 ℃

All-D Polymerase X variant V80A is-similar to the all-L polymerase X counterpart-if using thermal cycle extension, the entire 83-mer template It was observed that the strands could polymerize.

Example 7. Recombinant expression and purification of polymerases Dpo4 and Dpo4 variants composed of all L-amino acids

The polymerase Dpo4 was originally found in Sulfolobus Solfataricus (abbreviation Sso) (Boudsocq, 2001). The wild-type gene has an open reading frame (ORF) of 1,059 base pairs including a start codon and a stop codon. The encoded protein is 352 amino acids in length. This example describes a method in which polymerase Dpo4 and variants of polymerase Dpo4 are expressed in E. coli and purified using Strep-Tag.

7.1 Expression structure

Since the codon usage of Sso is different from that of E. coli, an E.coli-codon-optimized synthetic gene was purchased from GeneArt AG (Regensburg, Germany) for wild-type Sso polymerase Dpo4. This synthetic gene sequence was provided in the pENTRY-IBA10 vector (originator company: IBA GmbH, Goetingen, Germany). The codon-optimized open reading frame with a start codon but no stop codon had the following sequence:

AGCCATTGGCCTGGATAAATTTTTTGATACC.

To obtain the expression structure for polymerase Dpo4, also referred to as all-L polymerase Dpo4 (all-L polymerase Dpo4), pASG-IBA5 from pENTRY-IBA10 using a commercially available StarGate cloning kit (IBA GmbH). It was subcloned with a vector (IBA GmbH, Goetingen, Germany). Subcloning added a Strep-Tag II at the N-terminus, and a stop codon at the C-terminus and brought the gene under the control of the tet promoter. The structure was named pMJ343, and was used for the expression of all-L polymerase Dpo4 (all-L polymerase Dpo4) in E. coli. The pre-L-polymerase Dpo4 expressed from pMJ343 had the following 368 amino acid sequence:

MASAWSHPQFEKSGMIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDTGS.

The first 14 amino acids represented Strep-Tag II containing several spacer amino acids, the last two amino acids represented spacer amino acids, and the middle 352 amino acid portion was identical to the polymerase Dpo4 sequence found in Sso. did.

Expression structures for variants of all-L polymerase Dpo4 (all-L polymerase Dpo4) were made according to the manufacturer's protocol using a commercially available QuikChange kit (Stratagene GmbH, Waldbronn, Germany). Plasmid pMJ343 was used as a template. The oligonucleotides required for QuikChange are synthesized from NOXXON (QC_38_up, QC_38_low, QC_39_up, QC_39_low, QC_40_up, QC_40_low) or purchased from Purimex (Grebenstein, Germany) (QC_28_up, QC_lowup, QC_30_30_29) I did.

The following variant expression construct was created and used for the expression of a variant of all-L polymerase Dpo4 (all-L polymerase Dpo4) in E. coli:

Variant Expression structure Oligonucleotide structure used for the QuikChange mutagenesis procedure A155C pMJ361 QC_28_up.
(5'CAAAAATAAAGTGTTTGCCAAAATTGCATGCGATATGGCAAAACCG AATGGCATTAAAG 3')
QC_28_low
(5'CTTTAATGCCATTCGGTTTTGCCATATCGCATGCAATTTTGGCAAA CACTTTATTTTTG 3') V203C pMJ362 QC_29_up
(5'TGAAAAAACTGGGCATTAATAAACTGTGTGATACCCTGAGCATTGAATTTG 3')
QC_29_low
(5'CAAATTCAATGCTCAGGGTATCACACAGTTTATTAATGCCCAGTTTTTTCA 3') C31S pMJ363 QC_30_up
(5' TGAAAGGTAAACCGGTTGTTGTTTCTGTTTTTAGCGGTC 3')
QC_30_low
(5'GACCGCTAAAAACAGAAACAACAACCGGTTTACCTTTCA 3') A155C + V203C pMJ365 QC_28_up
QC_28_low
QC_29_up
QC_29_low S85C pMJ502 QC_38_up
(5'ATGCGCAAAGAAGTTTATCAGCAGGTTTGTAGCCGTATTATGAATC 3')
QC_38_low
(5'GATTCATAATACGGCTACAAACCTGCTGATAAACTTCTTTGCGCAT-3') S86C pMJ503 QC_39_up
(5'AAGTTTATCAGCAGGTTAGCTGTCGTATTATGAATCTGCTGCG 3')
QC_39_low
(5'CGCAGCAGATTCATAATACGACAGCTAACCTGCTGATAAACTT 3') S96C pMJ504 QC_40_up
(5'ATTATGAATCTGCTGCGCGAATATTGTGAAAAAATTGAAATTGCCAGCATT 3')
QC_40_low
(5'AATGCTGGCAATTTCAATTTTTTCACAATATTCGCGCAGCAGATTCATAAT 3')

7.2 Protein expression in E. coli

All-L polymerase Dpo4 (all-L polymerase Dpo4) was expressed in E. coli using the expression structure pMJ343. A mutant variant of all-L polymerase Dpo4 (all-L polymerase Dpo4) was expressed from pMJ361, pMJ362, pMJ363, pMJ365, pMJ502, pMJ503 or pMJ504. For expression, the E. coli strain'NEB express' (New England Biolabs, Frankfurt) using the appropriate expression structure'Transformation and Storage Solution' (Epicentre/Biozym, Hessisch Oldendorf, Germany). am Main, Germany), and maintained with the antibiotic Ampicillin. Expression was performed at 30° C. for 48 hours using Anhydrotetracyclin (IBA GmbH, Goetingen, Germany) as an inducer in'EnBase Flo'or'EnPresso' (BioSilta, Oulu, Finland) medium. Became. Cells were recovered by centrifugation and stored at -80°C or processed immediately.

7.3 Protein purification

Fresh or frozen E. coli cells were resuspended in'Buffer W'(100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) on ice, and'French Press''(G. Heinemann, Schwaeisch Gmuend, Germany) was lysed using a cell disruptor. Purification was performed at 4° C. using a'AKTA Express' system (GE healthcare, Freiburg, Germany) equipped with a 5 ml StrepTrap HP column. Step elution was performed using buffer W containing 2.5 mM Desthiobiotin (IBA GmbH, Goetingen, Germany). Fractions were analyzed using SDS-PAGE (Invitrogen, Karlsruhe, Germany), pooled and, if necessary, anion-ion in an'AKTA purifier' system (GE healthcare, Freiburg, Germany) equipped with a'Q HP' column. Further purification was performed by anion-ion-exchange chromatography. Protein identity was confirmed using LC-MS mass spectometry, the appropriate fractions were pooled, concentrated, and a VivaSpin 15R concentrator with a molecular weight 10,000 cut-off (MWCO) ( concentration devices) (VivaSciences/Sartorius Stedim Biotech, Goetingen, Germany) were used to re-buffered. The purified protein was stored at -20 °C in a buffer consisting of 100 mM KCl, 10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol. Protein concentration was measured by gel-densiometry and SYPRO Red (Invitrogen, Karlsruhe, Germany) staining using bovine serum albumin (abbreviated BSA) standard on SDS-PAGE.

Example 8. Production of synthetic polymerase Dpo4 consisting of two fragments

All-L polymerase Dpo4 (all-L polymerase Dpo4) has a length of 352 amino acids. In order to chemically produce pre-L-polymerase Dpo4, such synthetic pre-L-polymerase Dpo4 had to be assembled from shorter fragments that could be synthesized by solid-phase peptide synthesis, where the shorter fragment had a natural chemical reaction. It had to be linked by a peptide linkage method such as native chemical ligation. In this example, in order to obtain the synthetic pre-L-polymerase Dpo4, the ligation fragments 1-154, 155-352, 155-202 and 203-352 of Dpo4 were expressed in E. coli, purified, and natural Describe how they are connected to each other by chemical linkage.

8.1 Expression structure

As disclosed in Example 7, the gene for the pre-L-polymerase Dpo4 was obtained as a synthetic gene construct from a commercial source (GeneArt, Regensburg, Germany). All fragments of this example were cloned based on the codon-optimized sequence. The following expression structures were prepared for fragments 1-154, 155-352, 155-202 and 203-352 of the pre-L-polymerase Dpo4 variant A155C:

Fragment of Dpo4 (amino acid range) Expression structure 1-154-thioester pMJ370 155-352
Contains the A155C mutation pMJ384 203-352
Contains the A155C mutation pMJ385 155-202 thioester pMJ388

8.1.1 Fragment 1-154-thioester of all-L-polymerase Dpo4 variant A155C-Expression structure pMJ370

This structure is the Mxe GyrA intein and chitin-binding domain (CBD) which was used to produce the thioester following fragment 1-154 of the pre-L-polymerase Dpo4 variant A155C. Includes. The structure was assembled from two PCR products. PCR product 1 is a primer for amplifying fragment 1-154 of pMJ343 and all-L-polymerase Dpo4 variant A155C as a template MJ_1_90_DD (5'-Phosphate-AGCGGCTCTTCGATGATTGTGCTGTTTGTGGATTTT-3') and MJ_1_91_DD (5'-PGGCTCTAGGGGTT-3'-Phosphate-AGACT '). PCR product 2 as a template is pTWIN1 (New England Biolabs, Frankfurt am Main, Germany) and primers for amplifying Mxe Gyr A intein and CBD MJ_1_72_DD (5'-Phosphate-AGCGGCTCTTCGTGCATCACGGGAGAT-3') and MJ_1_73_DD (5 '-Phosphate-AGCGGCTCTTCGCCCTTGAAGCTGCCACAAGGCAGGAACGTT-3'). Primers MJ_1_90_DD and MJ_1_91_DD were purchased from Purimex (Grebenstein, Germany), and primers MJ_1_72_DD and MJ_1_73_DD were purchased from IBA GmbH (Goetingen, Germany). These two PCR products were gel-purified in a flash-gel system (LONZA, Basel, Switzerland) and pENTRY using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Goetingen, Germany). -Cloneed together in IBA20 to obtain pMJ366. Subcloning from pMJ366 in pASG-IBAwt1 (IBA GmbH, Goetingen, Germany) using a StarGate Transfer cloning kit (IBA GmbH, Goetingen, Germany) gave pMJ370. The pMJ370 structure encodes a fragment 1-154-thioester of the pre-L-polymerase Dpo4 variant A155C having the following protein sequence of 154 amino acids (after intein cleavage/product thioester). ):

MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIA-thioester.

8.1.2 Fragment 155-352 of pre-L-polymerase Dpo4 variant A155C-expression structure pMJ384

This structure contains fragments 155-352 of the pre-L-polymerase Dpo4 variant A155C followed by a'Profinity eXact' tag. The'Profinity eXact' tag was used for purification and proteolytic cleavage. The structure was assembled from two PCR products. PCR product 1 is a primer for amplifying pPAL7 (Bio-Rad, Muechen, Germany) and'Profinity eXact' tag as a template MJ_1_99_DD (5'-Phosphate-AGCGGCTCTTCGATGGGAGGGAAATCAAACGGGGAA-3') and MJ_1_100_DD (5'-Phosphate-Phosphate- AGCGGCTCTTCGGCACAAAGCTTTGAAGAGCTTGTC-3'). PCR product 2 was used as a template, primer MJ_1_96_DD (5'-Phosphate-AGCGGCTCTTCGTGCGATATGGCAAAACCGAATGGCATTAAA-3') and MJ_1_97_DD (5'-Phosphate-CAGGCTAA) and MJ_1_97_DD (5'-Phosphate-CAGGCTAA) for amplifying the dpo4 fragment 155-352 containing the pMJ361 and A155C mutations I made it. Primers MJ_1_96_DD, MJ_1_97_DD, MJ_1_99_DD and MJ_1_100_DD were purchased from Purimex (Grebenstein, Germany). These two PCR products were gel-purified in a flash-gel system (LONZA, Basel, Switzerland) and pENTRY using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Goetingen, Germany). -Cloneed together in IBA20 to obtain pMJ382. Subcloning from pMJ382 in pASG-IBAwt1 (IBA GmbH, Goetingen, Germany) using a StarGate Transfer cloning kit (IBA GmbH, Goetingen, Germany) yielded pMJ384. The pMJ384 structure encodes a fragment 155-352 of the pre-L-polymerase Dpo4 variant A155C having the following protein sequence 198 amino acids in length (after proteolytic cleavage):

C DMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIGIVSRGRTFPHGISKETAYSESVVAVTEDLDIGIVSRGRTFPHGISKETAYSESVKLLQKILDERIGLDKIRRKLLQKILDER

8.1.3 Fragment 203-352 of all-L-polymerase Dpo4 variant A155C-expression structure pMJ385

This structure contains the dpo4 fragment 203-352 containing the mutation V203C followed by a'Profinity eXact' tag. The'Profinity eXact' tag was used for purification and proteolytic cleavage. The structure was assembled from two PCR products. PCR product 1 is a primer for amplifying pPAL7 (Bio-Rad, Muechen, Germany) and'Profinity eXact' tag as a template MJ_1_99_DD (5'-Phosphate-AGCGGCTCTTCGATGGGAGGGAAATCAAACGGGGAA-3') and MJ_1_100_DD (5'-Phosphate-Phosphate- AGCGGCTCTTCGGCACAAAGCTTTGAAGAGCTTGTC-3'). PCR product 2 uses primers MJ_1_98_DD (5'-Phosphate-AGCGGCTCTTCGTGTGATACCCTGAGCATTGAATTT-3') and MJ_1_97_DD (5'-Phosphate-AGCGGCTAATCGCTTAGCGGCT') to amplify the dpo4 fragment 203-352 containing the pMJ362 and V203C mutations as templates. I made it. Primers MJ_1_97_DD, MJ_1_98_DD, MJ_1_99_DD and MJ_1_100_DD were purchased from Purimex (Grebenstein, Germany). These two PCR products were gel-purified in a flash-gel system (LONZA, Basel, Switzerland) and pENTRY using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Goetingen, Germany). Cloning together in -IBA20 gave pMJ383. Subcloning from pMJ383 in pASG-IBAwt1 (IBA GmbH, Goetingen, Germany) using the StarGate Transfer cloning kit (IBA GmbH, Goetingen, Germany) gave pMJ385. The pMJ385 structure encodes the dpo4 fragment 203-352 V203C having the following protein sequence 150 amino acids long (after proteolytic cleavage):

C DTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT

8.1.4 Fragment 155-202 of pre-L-polymerase Dpo4-expression structure pMJ388

This structure contains the Mxe GyrA intein and chitin-binding domain (CBD), which was used to produce the thioester following the dpo4 fragment 155-202. The structure was assembled from two PCR products. PCR product 1 was made using primers MJ_1_101_DD (5'-Phosphate-AGCGGCTCTTCGATGGCAGATATGGCAAAACCGAAT-3') and MJ_1_102_DD (5'-Phosphate-AGCGGAACTTCGGCAGTTT-3'TG) for amplifying pMJ343 and dpo4 155-202 fragments as templates. PCR product 2 as a template is pTWIN1 (New England Biolabs, Frankfurt am Main, Germany) and primers for amplifying Mxe Gyr A intein and CBD MJ_1_72_DD (5'-Phosphate-AGCGGCTCTTCGTGCATCACGGGAGAT-3') and MJ_1_73_DD (5 '-Phosphate-AGCGGCTCTTCGCCCTTGAAGCTGCCACAAGGCAGGAACGTT-3'). Primers MJ_1_101_DD and MJ_1_102_DD were purchased from Purimex (Grebenstein, Germany), and primers MJ_1_72_DD and MJ_1_73_DD were purchased from IBA GmbH (Goetingen, Germany). These two PCR products were gel-purified in a flash-gel system (LONZA, Basel, Switzerland) and pENTRY using the StarGate Mutagenesis ENTRY cloning kit (IBA GmbH, Goetingen, Germany). -Cloneed together in IBA20 to obtain pMJ386. Subcloning from pMJ386 in pASG-IBAwt1 (IBA GmbH, Goetingen, Germany) using a StarGate Transfer cloning kit (IBA GmbH, Goetingen, Germany) yielded pMJ388. The pMJ388 structure encodes a dpo4 fragment 155-202 having the following protein sequence of 48 amino acids in length (after E. coli mediated cleavage of the starting methionine and after intein cleavage/product of thioester):

ADMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKL-thioester

8.2 Protein expression in E. coli

For expression, the E. coli strain'NEB express' (New England Biolabs, Frankfurt) using the appropriate expression structure'Transformation and Storage Solution' (Epicentre/Biozym, Hessisch Oldendorf, Germany). am Main, Germany), and maintained with the antibiotic Ampicillin. Expression was carried out at ambient temperature using 200 ng/ml Anhydrotetracyclin (IBA GmbH, Goetingen, Germany) as an inducer in'EnBase Flo'or'EnPresso' (BioSilta, Oulu, Finland) medium. ambient temperature) overnight. Cells were recovered by centrifugation and stored at -80°C or processed immediately.

8.3 Purification and production of thioesters from structures pMJ370 and pMJ388 with Mxe Gyr A intein

Fresh or frozen E. coli cells were resuspended in'column buffer' (20 mM HEPES, pH 8.5, 500 mM NaCl) on ice, and'French Press' (G. Heinemann, Schwaeisch Gmuend, Germany) was lysed using a cell disruptor. Purification was performed at 4° C. using the'AKTA Express' system (GE healthcare, Freiburg, Germany) equipped with a column containing chitin binding beads (New Englands Biolabs, Frankfurt am Main, Germany). After cell lysis and washing to baseline with column buffer, 50 mM 2-mercaptoethane sulfonate in column buffer to induce intein-mediated protein cleavage and thioester formation on the column (Abbreviation MESNA) and incubated at 4° C. for 20 hours. The digested protein carrying the thioester was washed on the column with a column buffer, concentrated, and then in a buffer consisting of 5 mM Bis-Tris, pH 6.5, 250 mM NaCl, in a buffer consisting of BioGel P60 medium material (BioRad , Muechen, Germany) was used to perform gel filtration. Protein concentration was measured by gel-densiometry and SYPRO Red (Invitrogen, Karlsruhe, Germany) staining using bovine serum albumin (BSA) standards on SDS-PAGE. Protein identity and the presence of thioesters were confirmed using LC-MS mass spectometry.

8.4 Purification and proteolytic cleavage from structure pMJ384 with'Profinity eXact' tag

Purification of fragment 155-352 of the pre-L-polymerase Dpo4 variant A155C from pMJ384 was carried out as follows: Fresh or frozen E. coli cells in'Buffer W'(100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) and lysed using a'French Press' (G. Heinemann, Schwaeisch Gmuend, Germany) cell disruptor. Purification was performed at 4° C. using a'AKTA Express' system (GE healthcare, Freiburg, Germany) equipped with a 5 ml StrepTrap HP column. Step elution was performed using buffer W containing 2.5 mM Desthiobiotin (IBA GmbH, Goetingen, Germany). The eluted protein was buffered in'Profinity eXact elution buffer' (0.1 M Na-phosphate, pH 7.2, 0.1 M NaF) using a HiPrep 26/10 desalting column (GE healthcare, Freiburg, Germany). After the exchange, it was slowly pumped through the Profinity eXact column. Concentrate the sample, supply Tris(2-carboxyethyl)phosphine)(TCEP) to a final concentration of 1 mM, and then HiLoad 16/60 developed in a buffer consisting of 5 mM Bis-Tris, pH 6.5, 250 mM NaCl. Gel filtration was applied using a Superdex 75 prep grade column (GE healthcare, Freiburg, Germany). The protein was concentrated and stored at -80 °C. Protein concentration was measured by gel-densiometry and SYPRO Red (Invitrogen, Karlsruhe, Germany) staining using bovine serum albumin (BSA) standards on SDS-PAGE. Protein identity was confirmed using LC-MS mass spectometry.

8.5 Purification and proteolytic cleavage from structure pMJ385 with'Profinity eXact' tag

Purification of the dpo4 fragment 203-352 V203C from pMJ385 was carried out as follows: inclusion bodies were prepared and as described in the'i-FOLD Protein refolding system' manual (Novagen/Merck-Millipore, Darmstadt, Germany). I changed it. Solubilized protein was exchanged with'Buffer W'(100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) using Sephadex G-25 fine material, and the StrepTrap HP column It was purified at 4°C using the'AKTA Express' system (GE healthcare, Freiburg, Germany) equipped with (GE healthcare, Freiburg, Germany). Step elution was performed using buffer W containing 2.5 mM Desthiobiotin (IBA GmbH, Goetingen, Germany). NaF was supplied to the eluted protein solution to a final concentration of 0.1 M, and Tris(2-carboxyethyl)phosphine) (TCEP) was supplied to a final concentration of 1 mM, and then slowly pumped through a Profinity eXact column. The flowthrough was diluted 1:3 with deionized water, and the pH was adjusted to 7.2 using HCl. Samples were subjected to cation-exchange chromatography using a HiTrap SP HP column (GE healthcare, Freiburg, Germany) equilibrated in'Buffer A'(50 mM Na-Phosphate, pH 7.2, 1 mM 2-mercaptoethanol). -chromatography). Fractions were collected, concentrated, and applied to gel filtration using a HiLoad 16/60 Superdex 75 prep grade column (GE healthcare, Freiburg, Germany) developed in deionized water. The developed HiLoad 16/60 Superdex 75 prep grade column (GE healthcare, Freiburg, Germany) was used to apply gel filtration. Proteins were flash frozen in liquid nitrogen and lyophilized. Protein concentration was measured by gel-densiometry and SYPRO Red (Invitrogen, Karlsruhe, Germany) staining using bovine serum albumin (BSA) standards on SDS-PAGE. Protein identity was confirmed using LC-MS mass spectometry.

8.6 Synthesis of Pre-L-polymerase Dpo4 Variant A155C by Native Chemical Ligation of Fragment 1-154-thioester and 155-352

Fragment 1-154-thioester and 155-352 of the all-L-polymerase Dpo4 variant A155C in 2% Triton X100, 1% thiophenol and 5 mM Tris It was dissolved at 50 μM in a TRIS-buffer (pH 8.6) containing (2-carboxyethyl)phosphine hydrochloride. The reaction mixture was shaken at room temperature for 72 hours. Next, the success of the connection was determined by SDS-PAGE (Fig. 11A) and LC-ESI-mass spectrometer (RP18-column, gradient of ACN having 0.1% TFA in water with 5-95% 0.1% TFA in 20 minutes, Fig. 11B). A clear band was found in the lane around 41 kDa indicating full length polymerase. The theoretical molecular weight (M _theor = 40223 Da) corresponds to the observed molecular weight (M _obs = 40265 Da) as seen by ESI-MS.

8.7 Synthesis of fragment 155-352 by natural chemical ligation of fragment 155-202-thioester and 203-352

Fragments of all-L-polymerase Dpo4 (all-L-polymerase Dpo4) 155-202-thioester and 203-352 V203C were mixed with 2% SDS, 1% thiophenol and 5 mM tris (2- It was dissolved at 0.2 M in a TRIS-buffer (pH 8.6) containing carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride). The reaction mixture was shaken at room temperature for 72 hours. Then, analysis of the success of the ligation was performed by SDS-PAGE (Figure 20A) and LC-ESI-mass spectrometer (RP18-column, gradient of ACN with 0.1% TFA in water with 5-95% 0.1% TFA in 20 min, This was done by Figure 20B). A clear band was found in lane 7 near 21.5 kDa indicating the ligation product. Theoretical molecular weight (M _theor = 22749 Da) corresponds to a molecular weight (M _obs = 22769 Da) observed seen by the ESI-MS.

Example 9. Activity of polymerase Dpo4 composed of L-amino acid and variants of polymerase Dpo4

This Example is for all-L-polymerase Dpo4 (all-L-polymerase Dpo4) and all-L-polymerase Dpo4 variants according to Example 7 and the synthetic pre-L-polymerase Dpo4 according to Example 8. The PCR activity test is described.

9.1 Template for PCR activity assay

The template for the PCR reaction is the 83-mer single-stranded D-DNA oligonucleotide (MJ_1_1_DD) and the opposite strand is made in the first thermal cycle. Thereafter, both strands serve as a template for exponential amplification. Template DNA oligonucleotides and DNA primers are synthesized in NOXXON in D-configuration.

List of oligonucleotides for PCR activity assay:

designation Nucleotide length Sequence (5'→3') MJ_1_1_DD 83 GTGGAACCGACAACTTGTGCTGCGTCCAGCATAAGAAAGGAGCTCCCTCAGAAGAAGCTGCGCAGCGTGCCAGTCTGAGCTCC DE4.40T7 38 TCTAATACGACTCACTATAGGAGCTCAGACTGGCACGC DE4.40R 20 GTGGAACCGACAACTTGTGC

9.2 PCR reaction

15 µl PCR reactions were each 0.2 mM of four D-dNTP's, 10 nM 83-mer ssDNA (MJ_1_1_DD) template, 1 μM forward and reverse primers, 1x ThermoPol buffer (Invitrogen, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8@25° C.) and at least 0.67 ng/µl of pre-L-polymerase Dpo4 or pre-L-polymerase Dpo4. Variant or synthetic pre-L-polymerase Dpo4 was included. The forward primer was DE4.40T7 and the reverse primer was DE4.40R, and a PCR product of 102 base pairs in length was obtained. D-dNTP's were purchased from Rovalab (Teltow, Germany). The negative control was performed by omitting the pre-L-polymerase Dpo4 or a variant of the pre-L-polymerase Dpo4 or the synthetic pre-L-polymerase Dpo4. Positive controls were performed using commercially available pre-L-dpo4 (New England Biolabs, Frankfurt am Main, Germany).

The thermal cycling program consisted of 1 cycle (85° C., 3 minutes), then at least 7 cycles (85° C., 30 seconds/56° C., 1 minute/60° C., 4 minutes), then holding at 4° C. 4° C. aliquots of the PCR reaction were mixed with the sample loading buffer and analyzed on TBE-PAGE or agarose gel (LONZA, Cologne, Germany). Among others, a DNA standard ladder comprising a 100 bp band was applied to the gel.

9.3 Activity check

All-L-polymerase Dpo4 and all-L-polymerase Dpo4 variants A155C, V203C, C31S, A155C/V203C, S85C, S86C, S96C, and synthetic pre-L-polymerase Dpo4 were tested. All polymerases tested were able to amplify the template strand in the PCR reaction. Figure 12A shows the analysis of PCR reactions performed using the variants A155C, V203C, C31S, A155C/V203C of the pre-L-polymerase Dpo4, all of which are about 100 bp compared to the DNA standard ladder. Bands in the range of. The size of the predicted PCR product was 102 base pairs. The band does not appear in the negative control, where the polymerase is omitted. In addition, the 102 bp band moves better than the 83-mer template. 12B shows the analysis of the PCR reaction performed with the synthetic pre-L-polymerase dpo4, which was made by ligation of the recombinant pre-L-polymerase dpo4 and the fragment. The gel showed a band in the range of about 100 bp when compared to a DNA standard ladder. The size of the predicted PCR product was 102 base pairs. The band is very weak in the negative control, where the polymerase is omitted. Recombinant pre-L-polymerase dpo4 and synthetic pre-L-polymerase dpo4 exhibit the same activity as shown when compared to lanes 2 and 3 from Figure 12B.

Example 10. Synthesis of a variant of the polymerase Dpo4 consisting of D-amino acid

Synthesis of the pre-D polymerase Dpo variant A155C/V203C within this example is described. I -D polymerase amino acid sequence of Dpo mutant A155C / V203C is Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIACDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLCDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-OH.

All amino acids used are protected according to the Solid-phase Peptide Synthesis Fmoc/tBu-strategy requirements ( Eric Atherton et al, 1981). All amino acids used are D-amino acids (Bachem, Bubendorf, Switzerland).

10.1 H-d-Met-OGp(Boc) ₂ Synthesis of

1 mmole Zd-Met-OH, 0.9 eq. TBTU and 0.9 mmol HO-Gp(Boc) ₂ were dissolved in 10 ml DMF. 2 eq. After adding DIPEA, the solution was stirred for 2 hours. After evaporation of the solvent, the raw product was purified by flash chromatography using DCM. The pure fractions of Zd-Met-OGp(Boc) ₂ were collected and the solvent was evaporated.

Zd-Met-OGp(Boc) ₂ was dissolved in 10 ml MeOH and washed with argon. Hydrolytic cleavage of the N-terminal Z-group was carried out for 2 hours by addition of a Pd/C catalyst and H ₂ . After Hd-Met-OGp(Boc) ₂ was filtered, MeOH was evaporated under reduced pressure. Analysis was carried out using reverse phase HPLC and mass spectrometry. The calculated mass of 482 Da is consistent with the measured mass of 483 Da.

10.2 Fully protected all-D-peptide H-RTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT-NH ₂ (One) Synthesis of

0.1 mmole Fmoc-Sieber rink amide NovaSynTG resin (0.1 mmole Fmoc-Sieber rink amide NovaSynTG resin) was added to 5 eq. in 6 ml NMP for 45 min. Amino acids, eq. 4.9 eq. HATU and 10 eq. Fmoc-d-Thr(tBu)-OH was deprotected using DIPEA before loading.

Automatic synthesis was performed using ABI 433 with the FASTmoc protocol. 10 eq. of amino acids was added to 9 eq. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP.

Cleavage of the fully protected peptide acid was carried out by incubating the peptidyl resin twice in 10 ml of 1% (v/v) TFA in DCM for 2 hours. After filtering the peptide, the solvent was evaporated and the residue was precipitated using ice-cold diethyl ether. The precipitated peptide was separated and dried.

The final product was characterized by HPLC and mass spectrometry (Figure 13). The calculated mass of the product (6244 Da) corresponds to the measured mass (6249 Da).

10.3 Fully Protected Pre-D-Peptide Boc-VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRG-OH (2) Synthesis of

0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-d-Gly-OH as described in Barlos et al. (Barlos et al., 1989). Therefore, 0.10 mmol resin was incubated twice for 30 minutes with 0.6 mmole thionylchloride and then washed with DCM. Then, the resin was incubated in 6 ml DCM with 0.6 mmole Fmoc-Gly-OH and 2.4 mmol DIPEA for 90 minutes. Then, the resin was blocked three times for 10 minutes using 10% MeOH (v/v), 10% DIPEA (v/v) solution in DCM and washed with DCM. Automatic synthesis was performed using ABI 433 with the FASTmoc protocol. 10 eq. of amino acids was added to 9 eq. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP. Double coupling was performed after coupling of 39 amino acids.

Cleavage of the fully protected peptide acid was performed by incubating the peptidyl resin twice in 10 ml of 30% (v/v) HFIP in DCM for 2 hours. After filtering the peptide, the solvent was evaporated and the residue was precipitated using ice-cold diethyl ether. The precipitated peptide was separated and dried. The final product was characterized by HPLC and mass spectrometry (Figure 14). The experimentally determined mass of the product (11289 Da) is consistent with the theoretical value (11286 Da).

10.4 all-D-peptide H-VDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGR IVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDEELFRAIEESYYKLDKRIPKAIHVVAVTEDLDEELFRAIEESYYKLDKRIPKAIHVVAVTEDLDEELFRAIEESYYKLDKRIPKAIHVVAVTEDLDEELFRAIEESYYKLDKRIPKAIHVVAVTEDLDKISRGRKTFKDTYS by condensation of fragments of peptide 1 by condensation ₂ (3) Synthesis of

5 μmole (2) and 1 eq. (1) Dissolved in 25% (v/v) TFE in DCM. 5 eq. PyBOP and 10 eq. After DIPEA was added, the mixture was stirred overnight. After evaporation of the solvent, the peptide was precipitated using ice cold diethyl ether and filtered.

Cleavage of the side chain protection groups of the peptide was performed with 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. Following evaporation of TFA the peptide was precipitated using ice cold diethyl ether. Reverse phase HPLC purification of the N-terminal Fmoc-protected peptide was performed on a C18 column using an ACN/water gradient. Fractions containing the product were collected and freeze-dried.

The final product was characterized by HPLC and mass spectrometry (FIG. 15). The experimentally determined mass of the product (17531 Da) corresponds to the theoretical molecular weight (17512 Da).

10.5 Pre-D-peptide Z-CDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKL-benzyl-thioester (4) Synthesis of

0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-D-Leu-OH as described in Barlos et al. (Barlos et al., 1989). Therefore, 0.10 mmol resin was incubated twice for 30 minutes with 0.6 mmole thionylchloride and then washed with DCM. Then, the resin was incubated in 6 ml DCM with 0.6 mmole Fmoc-d-Leu-OH and 2.4 mmol DIPEA for 90 minutes. Then, the resin was blocked three times for 10 minutes using a 0% MeOH (v/v), 10% DIPEA (v/v) solution in DCM and washed with DCM. Automatic synthesis was performed using ABI 433 with the FASTmoc protocol. 10 eq. of amino acids was added to 9 eq. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP.

Cleavage of the fully protected peptide acid was performed by incubating the peptidyl resin twice in 10 ml of 30% (v/v) HFIP in DCM for 2 hours. After filtering the peptide, the solvent was evaporated and the residue was precipitated using ice-cold diethyl ether. The precipitated peptide was separated and dried.

The N-terminal Z- and fully side chain protected peptide 4 was dissolved in DMF at 1 mM. 5 eq. PyBOP, 10 eq. DIPEA and 30 eq. After adding benzyl mercaptan, the mixture was stirred for 4 hours. Then DMF was evaporated, the peptide was precipitated and washed with ice cold diethyl ether. Side chain protecting groups were removed by treatment with 2.5% EDT, 2.5% water, and 2.5% TIS in TFA for 2 hours. After evaporation of TFA, the peptide was precipitated and washed with ice cold diethyl ether. Then, the peptide-benzyl-thioester was purified by reverse-phase HPLC and analyzed by reverse-phase HPLC (Fig. 16A) and ESI-MS (Fig. 16B). The theoretical molecular weight (M _theor = 5527 Da) corresponds to the observed molecular weight (M _obs = 5533 Da) as shown by ESI-MS.

10.6 Ex-D-peptide H-RKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIA-SMe ( 7 ) Synthesis

0.10 mmole TentaGel-R-NH ₂ resin in 6 ml NMP for 45 minutes at 5 eq. amino acid, eq. 4.9 eq. HATU and 10 eq. Loaded with Fmoc-d-Ala-OH using DIPEA. The resin was then washed with THF. Conversion of Fmoc-D-Ala-ψ[CS-NH]-R-TentaGel to 4 eq in THF at 80° C. for 2 hours. This was done by incubation with Lawesson's reagent. Subsequently, the resin was washed with NMP. The resin thus prepared was then activated as described above (ABI 433, FASTmoc protocol, 10 eq. amino acids were activated using 9 eq. HATU and 20 eq. DIPEA in NMP; the coupling time was 45 minutes and , Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP) used for automatic peptide synthesis. A double coupling step was performed after 44 amino acids joined.

Subsequently, the corresponding thioester was produced by incubating with methyl iodide in DMF overnight according to Sharma et al. (Sharma et al, 2011). After filtering the resin, the peptide thioester containing the solvent was evaporated and the residue was precipitated with ice-cold diethyl ether. Cleavage of side chain protection groups was performed with 2.5% EDT, 2.5% water, 2.5% TIS in TFA for 2 hours. Following evaporation of TFA, the peptide was precipitated with ice cold diethyl ether. Reverse phase HPLC purification of the peptide thioester was performed on a C18 column using an ACN/water gradient. Fractions containing the product were collected and freeze-dried.

The final product was characterized by HPLC (Fig. 17A) and mass spectrometry (Fig. 17B). The molecular weight (9155 Da) of the product measured by mass spectrometry was consistent with the calculated mass (9150 Da).

10.7 Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIV EAKKILPNAVYLPM-OGp ( 6 ) Synthesis

0.10 mmole TentaGel-R-Trityl resin was loaded with Fmoc-D-Gly-OH as described in Barlos et al. (Barlos et al., 1989). Therefore, 0.10 mmol resin was incubated twice for 30 minutes with 0.6 mmole thionylchloride and then washed with DCM. Subsequently, the resin was incubated in 6 ml DCM with 0.6 mmole Fmoc-D-Pro-OH and 2.4 mmol DIPEA for 90 minutes. Then, the resin was blocked three times for 10 minutes using 10% MeOH (v/v), 10% DIPEA (v/v) solution in DCM and washed with DCM. Automatic synthesis was performed using ABI 433 with the FASTmoc protocol. 10 eq. of amino acids was added to 9 eq. HATU and 20 eq. It was activated using DIPEA. The coupling time was 45 minutes, and Fmoc-deprotection was performed three times for 7 minutes with 20% (v/v) piperidine in NMP. Double coupling was performed after the coupling of 46 amino acids. N-terminal acetylation was performed three times for 10 minutes in DMF with 10% (v/v) acetic anhydride 9acetic anhydride) and 10% (v/v) DIPEA.

Cleavage of the fully protected peptide acid was performed by incubating the peptidyl resin twice in 10 ml of 30% (v/v) HFIP in DCM for 2 hours. After filtering the peptide, the solvent was evaporated and the residue was precipitated using ice-cold diethyl ether. The precipitated peptide was separated and dried.

0.01 mmole of fully protected peptide, 4 eq. PyBOP and 5 eq. Hd-Met-OGp(Boc) ₂ was dissolved in 6 ml NMP. 10 eq. After DIPEA was added, the mixture was stirred for 4 hours. Following this, the solvent was evaporated under reduced pressure and the residue was precipitated with ice cold diethyl ether. The precipitated peptide ester was dried and then the protection groups were cleaved in TFA for 2 hours using 2.5% EDT, 2.5% water, 2.5% TIS. Following evaporation of TFA, the peptide was precipitated with ice cold diethyl ether. Reverse phase HPLC purification of the peptide ester was performed on a C18 column using an ACN/water gradient. Fractions containing the product were collected and freeze-dried.

The final product was characterized by HPLC (Figure 18A) and mass spectrometry (Figure 18B). The experimentally determined mass (8547 Da) corresponds to the theoretical molecular weight (8541 Da).

10.8 peptide 3 Peptide having 4 All-D-peptide H-CDMAKPNGIKVIDDEEVKRLIRELDIADVPGIGNITAEKLKKLGINKLCDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSPHVARNLEYYKPYKPYLFKRNTFRNLEYYKPYKPYLFKDT (5) Synthesis of

Peptides 3 and 4 were all in a 0.2 M TRIS-buffer containing 2% SDS, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride). pH 8.6). The reaction mixture was shaken at room temperature for 72 hours. The mixture is then purified by reverse phase HPLC. To remove the N-terminal Z-protecting group, the peptide was added to 270 eq. TFA and 50 eq. Dissolve in thioanisol and shake at room temperature for 6 hours (Yoshiaki Kiso et al, 1980). After evaporation of TFA, the peptide was precipitated, washed with ice cold diethyl ether and purified again by reverse phase HPLC (Phenomenex, Aschaffenburg, Germany). Analysis of the free peptide 5 was performed by SDS-PAGE, reversed-phase UPLC and ESI-mass spectrometry. Find the exact mass of the product.

10.9 peptide 7 Peptide having 6 All-D-peptide Ac-MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVKAGIPIVEAKKILPNAVYLPMRKEVYQVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSYNASSRIMNLKEYNKREADY (8) Synthesis of

Peptide 6 was dissolved to 0.2 mM in sodium-phosphate buffer (100 mM, pH 8.5, and 100 mM NaCl included) containing 4 M Urea, and peptide 7 was dissolved to 0.6 mM. After the addition of 20 μM clostripain (Endoprotease Arg-C, Worthington Biochemical Corporation, Lakewood, NJ, USA), the reaction mixture was shaken at 37° C. overnight. The precipitated peptide was centrifuged, dissolved in H ₂ O/formic acid 80/20, and used RP-18-column (Phenomenex, Aschaffenburg, Germany) having a gradient of ACN in water of 5% to 95% within 30 minutes. Purified by reverse phase HPLC. The final peptide was analyzed by SDS-PAGE (Fig. 19A) and ESI-mass spectrometry (Fig. 19B). The band was found in lane 1 between 14,4 kDa and 21.5 kDa indicating the ligation product. The theoretical molecular weight of the product connection (M _theor = 17476 Da) corresponds to a molecular weight (M _obs = 17486 Da) observed seen by the ESI-MS.

10.10 peptide 5 Peptide having 8 Synthesis of all-D polymerase Dpo variant A115C/V203C by native chemical ligation of

0.2 M TRIS-buffer containing both peptides 5 and 8 in 2% Triton X100, 1% thiophenol and 5 mM tris(2-carboxyethyl)phosphine hydrochloride (pH 8.6). The reaction mixture was shaken at room temperature for 72 hours. The mixture was then purified by reverse phase HPLC and analyzed by SDS-PAGE, reverse phase UPLC and ESI-mass spectrometry. Find the exact mass of the product.

Example 11. Confirmation of the activity of the synthetic polymerase dpo4 consisting of D-amino acid

This example describes the PCR activity test for the pre-D polymerase Dpo4 variant A155C/V203C according to Example 10.

Surprisingly, the synthetic pre-D polymerase Dpo4 variant A155C/V203C was active without any additional refolding effort and is therefore used without further refolding procedures. The protein concentration was determined by a standard series of known proteins according to SYPRO-RED staining (Invitrogen, Karlsruhe, Germany) and pre-cast gels using a densiometric band on a BioRad Fx scanner instrument (Invitrogen, Karlsruhe. , Germany) on SDS-PAGE.

11.1 Template for PCR activity assay

The template for the PCR reaction is the 83-mer single-stranded L-DNA oligonucleotide (MJ_1_105_LD) and the opposite strand is made in the first thermal cycle. Thereafter, both strands serve as a template for exponential amplification. Template DNA oligonucleotides and DNA primers are synthesized in NOXXON in L-configuration.

List of oligonucleotides for PCR activity assay:

designation Nucleotide length Arrangement
(Configuration) Sequence (5'→3') MJ_1_105_LD 83 L GTGGAACCGACAACTTGTGCTGCGTCCAGCATAAGAAAGGAGCTCCCTCAGAAGAAGCTGCGCAGCGTGCCAGTCTGAGCTCC MJ_oligo_187_LD 38 L TCTAATACGACTCACTATAGGAGCTCAGACTGGCACGC MJ_oligo_189_LD 20 L GTGGAACCGACAACTTGTGC

11.2 PCR reaction

15 µl PCR reactions were each 0.2 mM of four L-dNTP's, 10 nM 83-mer ssDNA (MJ_1_105_LD) template, 1 μM forward and reverse primers, 1x ThermoPol buffer (Invitrogen, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8@25° C.) and at least 0.67 ng/ul of the pre-D-polymerase Dpo4 variant A155C/V203C. The forward primer is MJ_oligo_187_LD, which yields a 102 bp PCR product, distinguishable from the 83-mer template. The reverse primer is MJ_oligo_189_LD. L-dNTP's is

A PCR product of DE4.40R and 102 base pairs in length was obtained. D-dNTP's were purchased from Rovalab (Teltow, Germany). The negative control was performed by omitting the pre-L-polymerase Dpo4 or a variant of the pre-L-polymerase Dpo4 or the synthetic pre-L-polymerase Dpo4. Positive controls were performed using commercially available pre-L-dpo4 (New England Biolabs, Frankfurt am Main, Germany). L-dNTP's is owned by Rasayan, Inc. (Encinitas, CA, USA) was purchased by custom synthesis.

A negative control was performed without omitting the pre-D polymerase Dpo4 variant A155C/V203C.

The thermal cycling program consisted of 1 cycle (85° C., 3 minutes), then at least 7 cycles (85° C., 30 seconds/56° C., 1 minute/60° C., 4 minutes), then holding at 4° C. 4 °C aliquots of the PCR reaction were mixed with sample loading buffer and analyzed on a TBE gel. Among others, a DNA standard ladder comprising a 100 bp band was applied to the gel.

11.3 Activity check

PCR reactions with all-D polymerase Dpo4 variants A155C/V203C and L-DNA substrate and L-nucleotide yielded a band in the range of about 100 bp compared to a DNA standard ladder. The band does not appear in the negative control, where the polymerase is omitted. In addition, the 102 bp migrates better than the 83-mer template. Therefore, the pre-D polymerase Dpo4 variant A155C/V203C, which depends on the appearance of the L-DNA amplification product, confirms the activity of the synthetic pre-D polymerase Dpo4 variant A155C/V203C in the heat amplification process.

Example 12. Synthesis of D- or L-nucleic acid

ABI 394 synthesizer using 2'TBDMS DNA phosphoramidite chemistry (Damha and Ogilvie, 1993) with L-DNA nucleic acid or D-DNA nucleic acid with standard exocyclic amine protecting group (Applied Biosystems, Foster City) , CA, USA) by solid-phase synthesis. For DNA synthesis, dA(N-Bz)-, dC(N-Ac)-, dG(N-ibu)-, and dT of D- and L-configuration were applied. All phosphoramidites were purchased from ChemGenes, Wilmington, MA. After synthesis and deprotection, L-DNA nucleic acids or D-DNA nucleic acids were purified by gel electrophoresis.

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Features of the present invention disclosed in the specification, claims, and/or drawings may be materials for realizing the present invention in various forms separately and in combination thereof.

SEQUENCE LISTING <110> NOXXON Pharma AG <120> Enzymatic synthesis of L-nucleic acids <130> N 10099 PCT <140> PCT/EP2013/001458 <141> 2013-05-16 <150> EP 12 003 887.2 <151> 2012-05-16 <160> 88 <170> PatentIn version 3.3 <210> 1 <211> 174 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X <400> 1 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Val 65 70 75 80 Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 85 90 95 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 100 105 110 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile Arg Ile Arg Ala 115 120 125 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 130 135 140 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 145 150 155 160 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 165 170 <210> 2 <211> 174 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X variant <400> 2 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Gly 65 70 75 80 Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 85 90 95 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 100 105 110 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile Arg Ile Arg Ala 115 120 125 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 130 135 140 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 145 150 155 160 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 165 170 <210> 3 <211> 174 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X variant <400> 3 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Ala 65 70 75 80 Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 85 90 95 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 100 105 110 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile Arg Ile Arg Ala 115 120 125 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 130 135 140 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 145 150 155 160 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 165 170 <210> 4 <211> 174 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X variant <400> 4 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Val 65 70 75 80 Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 85 90 95 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 100 105 110 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Gly Arg Ile Arg Ala 115 120 125 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 130 135 140 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 145 150 155 160 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 165 170 <210> 5 <211> 174 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X variant <400> 5 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Val 65 70 75 80 Cys Gly Glu Arg Lys Ser Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 85 90 95 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 100 105 110 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile Arg Ile Arg Ala 115 120 125 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 130 135 140 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 145 150 155 160 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 165 170 <210> 6 <211> 17 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(2) <223> D-nucleotides <220> <221> misc_feature <222> (3)..(17) <223> L-nucleotides <400> 6 gggatcacag tgagtac 17 <210> 7 <211> 17 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(17) <223> L-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 7 gtaaaacgac ggccagt 17 <210> 8 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(33) <223> L-nucleotides <400> 8 actggccgtc gttttacagt actcactgtg atc 33 <210> 9 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(33) <223> L-nucleotides <400> 9 actggccgtc gttttaccgt actcactgtg atc 33 <210> 10 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(33) <223> L-nucleotides <400> 10 actggccgtc gttttacggt actcactgtg atc 33 <210> 11 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(33) <223> L-nucleotides <400> 11 actggccgtc gttttactgt actcactgtg atc 33 <210> 12 <211> 17 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(2) <223> D-nucleotides <220> <221> misc_feature <222> (3)..(17) <223> L-nucleotides <400> 12 gggatcacag tgagtac 17 <210> 13 <211> 12 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(12) <223> L-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 13 acgacggcca gt 12 <210> 14 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(33) <223> L-nucleotides <400> 14 actggccgtc gttctattgt actcactgtg atc 33 <210> 15 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 <400> 15 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 16 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 16 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Cys Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 17 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polmyerase Dpo4 variant <400> 17 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Cys Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 18 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 18 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Cys Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Cys Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 19 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 19 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Ser Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 20 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 20 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Cys Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 21 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 21 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Cys Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 22 <211> 352 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4 variant <400> 22 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Cys 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro 145 150 155 160 Asn Gly Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg 165 170 175 Glu Leu Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu 180 185 190 Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile 195 200 205 Glu Phe Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr 210 215 220 Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg 225 230 235 240 Val Arg Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg 245 250 255 Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser 260 265 270 Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala 275 280 285 Val Thr Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His 290 295 300 Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln 305 310 315 320 Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg 325 330 335 Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr 340 345 350 <210> 23 <211> 83 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(83) <223> L-nucleotides <400> 23 gtggaaccga caacttgtgc tgcgtccagc ataagaaagg agctccctca gaagaagctg 60 cgcagcgtgc cagtctgagc tcc 83 <210> 24 <211> 38 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(38) <223> L-nucleotides <400> 24 tctaatacga ctcactatag gagctcagac tggcacgc 38 <210> 25 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(20) <223> L-nucleotides <400> 25 gtggaaccga caacttgtgc 20 <210> 26 <211> 528 <212> DNA <213> African swine fever virus <220> <221> misc_feature <223> Polymerase X, E.coli codon optimized <400> 26 atgctgaccc tgattcaggg caaaaaaatc gtgaaccatc tgcgtagccg tctggccttt 60 gaatataacg gccagctgat taaaattctg agcaaaaaca ttgtggcggt gggcagcctg 120 cgtcgtgaag aaaaaatgct gaacgatgtg gatctgctga ttattgtgcc ggaaaaaaaa 180 ctgctgaaac atgtgctgcc gaacattcgt attaaaggcc tgagctttag cgtgaaagtg 240 tgcggcgaac gtaaatgcgt gctgtttatc gaatgggaaa aaaaaaccta ccagctggac 300 ctgtttaccg cgctggccga agaaaaaccg tatgcgatct ttcattttac cggtccggtg 360 agctatctga ttcgtattcg tgcggcgctg aaaaaaaaaa actacaaact gaaccagtat 420 ggcctgttta aaaaccagac cctggtgccg ctgaaaatta ccaccgaaaa agaactgatt 480 aaagaactgg gctttaccta tcgcattccg aaaaaacgcc tgtaataa 528 <210> 27 <211> 210 <212> PRT <213> African swine fever virus <220> <221> MISC_FEATURE <223> Polymerase X, His-tagged <400> 27 Met Arg Gly Ser His His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Arg Trp Gly Ser Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn 35 40 45 His Leu Arg Ser Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys 50 55 60 Ile Leu Ser Lys Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu 65 70 75 80 Lys Met Leu Asn Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys 85 90 95 Leu Leu Lys His Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe 100 105 110 Ser Val Lys Val Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp 115 120 125 Glu Lys Lys Thr Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu 130 135 140 Lys Pro Tyr Ala Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile 145 150 155 160 Arg Ile Arg Ala Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr 165 170 175 Gly Leu Phe Lys Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu 180 185 190 Lys Glu Leu Ile Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys 195 200 205 Arg Leu 210 <210> 28 <211> 34 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 28 tccggtgagc tatctgggtc gtattcgtgc ggcg 34 <210> 29 <211> 34 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 29 cgccgcacga atacgaccca gatagctcac cgga 34 <210> 30 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 30 tgagctttag cgtgaaaggg tgcggcgaac g 31 <210> 31 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 31 cgttcgccgc accctttcac gctaaagctc a 31 <210> 32 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 32 tgagctttag cgtgaaagcg tgcggcgaac g 31 <210> 33 <211> 31 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 33 cgttcgccgc acgctttcac gctaaagctc a 31 <210> 34 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 34 tgaaagtgtg cggcgaacgt aaaagcgtgc tgttta 36 <210> 35 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 35 taaacagcac gcttttacgt tcgccgcaca ctttca 36 <210> 36 <211> 15 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 36 gatcacagtg agtac 15 <210> 37 <211> 17 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 37 gtaaaacgac ggccagt 17 <210> 38 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 38 actggccgtc gttttacagt actcactgtg atc 33 <210> 39 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 39 actggccgtc gttttaccgt actcactgtg atc 33 <210> 40 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 40 actggccgtc gttttacggt actcactgtg atc 33 <210> 41 <211> 33 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 41 actggccgtc gttttactgt actcactgtg atc 33 <210> 42 <211> 12 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 42 acgacggcca gt 12 <210> 43 <211> 40 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> A is AcM <220> <221> MISC_FEATURE <222> (40)..(40) <223> L is L-OGp <400> 43 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu 35 40 <210> 44 <211> 39 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> R is H-R <220> <221> MISC_FEATURE <222> (39)..(39) <223> A is A-SMe <400> 44 Arg Arg Glu Glu Lys Leu Asn Asp Val Asp Leu Leu Ile Ile Val Pro 1 5 10 15 Glu Lys Lys Leu Leu Lys His Val Leu Pro Asn Ile Arg Ile Lys Gly 20 25 30 Leu Ser Phe Ser Val Lys Ala 35 <210> 45 <211> 94 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> C is H-C <220> <221> MISC_FEATURE <222> (94)..(94) <223> L is L-OH <400> 45 Cys Gly Glu Arg Lys Cys Val Leu Phe Ile Glu Trp Glu Lys Lys Thr 1 5 10 15 Tyr Gln Leu Asp Leu Phe Thr Ala Leu Ala Glu Glu Lys Pro Tyr Ala 20 25 30 Ile Phe His Phe Thr Gly Pro Val Ser Tyr Leu Ile Arg Ile Arg Ala 35 40 45 Ala Leu Lys Lys Lys Asn Tyr Lys Leu Asn Gln Tyr Gly Leu Phe Lys 50 55 60 Asn Gln Thr Leu Val Pro Leu Lys Ile Thr Thr Glu Lys Glu Leu Ile 65 70 75 80 Lys Glu Leu Gly Phe Thr Tyr Arg Ile Pro Lys Lys Arg Leu 85 90 <210> 46 <211> 80 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> M is Ac-M <220> <221> MISC_FEATURE <222> (80)..(80) <223> A is A-SMe <400> 46 Met Leu Thr Leu Ile Gln Gly Lys Lys Ile Val Asn His Leu Arg Ser 1 5 10 15 Arg Leu Ala Phe Glu Tyr Asn Gly Gln Leu Ile Lys Ile Leu Ser Lys 20 25 30 Asn Ile Val Ala Val Gly Ser Leu Arg Arg Glu Glu Lys Met Leu Asn 35 40 45 Asp Val Asp Leu Leu Ile Ile Val Pro Glu Lys Lys Leu Leu Lys His 50 55 60 Val Leu Pro Asn Ile Arg Ile Lys Gly Leu Ser Phe Ser Val Lys Ala 65 70 75 80 <210> 47 <211> 19 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Fluorescent dye Atto-532 attached <400> 47 ggagctcaga ctggcacgc 19 <210> 48 <211> 83 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 48 gtggaaccga caacttgtgc tgcgtccagc ataagaaagg agctccctca gaagaagctg 60 cgcagcgtgc cagtctgagc tcc 83 <210> 49 <211> 21 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <222> (1)..(2) <223> D-nucleotides <220> <221> misc_feature <222> (3)..(21) <223> L-nucleotides <400> 49 ggggagctca gactggcacg c 21 <210> 50 <211> 1056 <212> DNA <213> Sulfolobus solfataricus <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <223> Polymerase Dpo4, E.coli codon optimized <400> 50 atgattgtgc tgtttgtgga ttttgattat ttttatgccc aggtggaaga agttctgaat 60 ccgagcctga aaggtaaacc ggttgttgtt tgtgttttta gcggtcgctt tgaagatagc 120 ggtgcagttg caaccgccaa ttatgaagcc cgtaaatttg gtgttaaagc cggtattccg 180 attgttgaag ccaaaaaaat tctgccgaat gcagtttatc tgccgatgcg caaagaagtt 240 tatcagcagg ttagcagccg tattatgaat ctgctgcgcg aatatagcga aaaaattgaa 300 attgccagca ttgatgaagc ctatctggat attagcgata aagtgcgcga ttatcgcgaa 360 gcatataatc tgggcctgga aattaaaaat aaaatcctgg aaaaagaaaa aattaccgtg 420 accgtgggca ttagcaaaaa taaagtgttt gccaaaattg cagcagatat ggcaaaaccg 480 aatggcatta aagtgattga tgatgaagaa gtgaaacgtc tgattcgcga actggatatt 540 gcagatgttc cgggtattgg caatattacc gcagaaaaac tgaaaaaact gggcattaat 600 aaactggttg ataccctgag cattgaattt gataaactga aaggcatgat tggtgaagcg 660 aaagccaaat atctgattag cctggcacgt gatgaatata atgaaccgat tcgtacccgt 720 gttcgtaaaa gcattggtcg tattgtgacc atgaaacgca atagccgtaa tctggaagaa 780 attaaaccgt acctgtttcg tgcaattgaa gaaagctatt ataaactgga taaacgcatt 840 ccgaaagcca ttcatgttgt tgcagttacc gaagatctgg atattgttag ccgtggtcgt 900 acctttccgc atggtattag caaagaaacc gcctatagcg aaagcgttaa actgctgcag 960 aaaatcctgg aagaagatga acgtaaaatt cgtcgtattg gtgtgcgctt tagcaaattt 1020 attgaagcca ttggcctgga taaatttttt gatacc 1056 <210> 51 <211> 368 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4, Strep-tagged <400> 51 Met Ala Ser Ala Trp Ser His Pro Gln Phe Glu Lys Ser Gly Met Ile 1 5 10 15 Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu Glu Val 20 25 30 Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val Phe Ser 35 40 45 Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr Glu Ala 50 55 60 Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala Lys Lys 65 70 75 80 Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val Tyr Gln 85 90 95 Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser Glu Lys 100 105 110 Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser Asp Lys 115 120 125 Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile Lys Asn 130 135 140 Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile Ser Lys 145 150 155 160 Asn Lys Val Phe Ala Lys Ile Ala Ala Asp Met Ala Lys Pro Asn Gly 165 170 175 Ile Lys Val Ile Asp Asp Glu Glu Val Lys Arg Leu Ile Arg Glu Leu 180 185 190 Asp Ile Ala Asp Val Pro Gly Ile Gly Asn Ile Thr Ala Glu Lys Leu 195 200 205 Lys Lys Leu Gly Ile Asn Lys Leu Val Asp Thr Leu Ser Ile Glu Phe 210 215 220 Asp Lys Leu Lys Gly Met Ile Gly Glu Ala Lys Ala Lys Tyr Leu Ile 225 230 235 240 Ser Leu Ala Arg Asp Glu Tyr Asn Glu Pro Ile Arg Thr Arg Val Arg 245 250 255 Lys Ser Ile Gly Arg Ile Val Thr Met Lys Arg Asn Ser Arg Asn Leu 260 265 270 Glu Glu Ile Lys Pro Tyr Leu Phe Arg Ala Ile Glu Glu Ser Tyr Tyr 275 280 285 Lys Leu Asp Lys Arg Ile Pro Lys Ala Ile His Val Val Ala Val Thr 290 295 300 Glu Asp Leu Asp Ile Val Ser Arg Gly Arg Thr Phe Pro His Gly Ile 305 310 315 320 Ser Lys Glu Thr Ala Tyr Ser Glu Ser Val Lys Leu Leu Gln Lys Ile 325 330 335 Leu Glu Glu Asp Glu Arg Lys Ile Arg Arg Ile Gly Val Arg Phe Ser 340 345 350 Lys Phe Ile Glu Ala Ile Gly Leu Asp Lys Phe Phe Asp Thr Gly Ser 355 360 365 <210> 52 <211> 59 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 52 caaaaataaa gtgtttgcca aaattgcatg cgatatggca aaaccgaatg gcattaaag 59 <210> 53 <211> 59 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 53 ctttaatgcc attcggtttt gccatatcgc atgcaatttt ggcaaacact ttatttttg 59 <210> 54 <211> 51 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 54 tgaaaaaact gggcattaat aaactgtgtg ataccctgag cattgaattt g 51 <210> 55 <211> 51 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 55 caaattcaat gctcagggta tcacacagtt tattaatgcc cagttttttc a 51 <210> 56 <211> 39 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 56 tgaaaggtaa accggttgtt gtttctgttt ttagcggtc 39 <210> 57 <211> 39 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 57 gaccgctaaa aacagaaaca acaaccggtt tacctttca 39 <210> 58 <211> 46 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 58 atgcgcaaag aagtttatca gcaggtttgt agccgtatta tgaatc 46 <210> 59 <211> 46 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 59 gattcataat acggctacaa acctgctgat aaacttcttt gcgcat 46 <210> 60 <211> 43 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 60 aagtttatca gcaggttagc tgtcgtatta tgaatctgct gcg 43 <210> 61 <211> 43 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 61 cgcagcagat tcataatacg acagctaacc tgctgataaa ctt 43 <210> 62 <211> 51 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 62 attatgaatc tgctgcgcga atattgtgaa aaaattgaaa ttgccagcat t 51 <210> 63 <211> 51 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 63 aatgctggca atttcaattt tttcacaata ttcgcgcagc agattcataa t 51 <210> 64 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 64 agcggctctt cgatgattgt gctgtttgtg gatttt 36 <210> 65 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 65 agcggctctt cggcatgcaa ttttggcaaa cacttt 36 <210> 66 <211> 27 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 66 agcggctctt cgtgcatcac gggagat 27 <210> 67 <211> 42 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 67 agcggctctt cgcccttgaa gctgccacaa ggcaggaacg tt 42 <210> 68 <211> 154 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4, fragment 1-154 <400> 68 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala 145 150 <210> 69 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 69 agcggctctt cgatgggagg gaaatcaaac ggggaa 36 <210> 70 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 70 agcggctctt cggcacaaag ctttgaagag cttgtc 36 <210> 71 <211> 42 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 71 agcggctctt cgtgcgatat ggcaaaaccg aatggcatta aa 42 <210> 72 <211> 40 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 72 agcggctctt cgcccttagg tatcaaaaaa tttatccagg 40 <210> 73 <211> 198 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4, fragment 155-352 <400> 73 Cys Asp Met Ala Lys Pro Asn Gly Ile Lys Val Ile Asp Asp Glu Glu 1 5 10 15 Val Lys Arg Leu Ile Arg Glu Leu Asp Ile Ala Asp Val Pro Gly Ile 20 25 30 Gly Asn Ile Thr Ala Glu Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu 35 40 45 Val Asp Thr Leu Ser Ile Glu Phe Asp Lys Leu Lys Gly Met Ile Gly 50 55 60 Glu Ala Lys Ala Lys Tyr Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn 65 70 75 80 Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile Val Thr 85 90 95 Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe 100 105 110 Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys 115 120 125 Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val Ser Arg 130 135 140 Gly Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu 145 150 155 160 Ser Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile 165 170 175 Arg Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu 180 185 190 Asp Lys Phe Phe Asp Thr 195 <210> 74 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 74 agcggctctt cgtgtgatac cctgagcatt gaattt 36 <210> 75 <211> 150 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4, fragment 155-352 <400> 75 Cys Asp Thr Leu Ser Ile Glu Phe Asp Lys Leu Lys Gly Met Ile Gly 1 5 10 15 Glu Ala Lys Ala Lys Tyr Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn 20 25 30 Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile Val Thr 35 40 45 Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe 50 55 60 Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys 65 70 75 80 Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val Ser Arg 85 90 95 Gly Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu 100 105 110 Ser Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile 115 120 125 Arg Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu 130 135 140 Asp Lys Phe Phe Asp Thr 145 150 <210> 76 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 76 agcggctctt cgatggcaga tatggcaaaa ccgaat 36 <210> 77 <211> 36 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <220> <221> misc_feature <222> (1)..(1) <223> Phosphate attached <400> 77 agcggctctt cggcacagtt tattaatgcc cagttt 36 <210> 78 <211> 48 <212> PRT <213> Sulfolobus solfataricus <220> <221> MISC_FEATURE <223> Polymerase Dpo4, fragment 155-202 <220> <221> MISC_FEATURE <222> (1)..(48) <223> L is L-thioester <400> 78 Ala Asp Met Ala Lys Pro Asn Gly Ile Lys Val Ile Asp Asp Glu Glu 1 5 10 15 Val Lys Arg Leu Ile Arg Glu Leu Asp Ile Ala Asp Val Pro Gly Ile 20 25 30 Gly Asn Ile Thr Ala Glu Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu 35 40 45 <210> 79 <211> 38 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 79 tctaatacga ctcactatag gagctcagac tggcacgc 38 <210> 80 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <220> <221> misc_feature <223> D-nucleotides <400> 80 gtggaaccga caacttgtgc 20 <210> 81 <211> 53 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> R is H-R <220> <221> MISC_FEATURE <222> (53)..(53) <223> T is T-NH2 <400> 81 Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu Ser 1 5 10 15 Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile Arg 20 25 30 Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu Asp 35 40 45 Lys Phe Phe Asp Thr 50 <210> 82 <211> 97 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> V is Boc-V <220> <221> MISC_FEATURE <222> (97)..(97) <223> G is G-OH <400> 82 Val Asp Thr Leu Ser Ile Glu Phe Asp Lys Leu Lys Gly Met Ile Gly 1 5 10 15 Glu Ala Lys Ala Lys Tyr Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn 20 25 30 Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile Val Thr 35 40 45 Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe 50 55 60 Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys 65 70 75 80 Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val Ser Arg 85 90 95 Gly <210> 83 <211> 150 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> V is H-V <220> <221> MISC_FEATURE <222> (150)..(150) <223> T is T-NH2 <400> 83 Val Asp Thr Leu Ser Ile Glu Phe Asp Lys Leu Lys Gly Met Ile Gly 1 5 10 15 Glu Ala Lys Ala Lys Tyr Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn 20 25 30 Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile Val Thr 35 40 45 Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe 50 55 60 Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys 65 70 75 80 Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val Ser Arg 85 90 95 Gly Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu 100 105 110 Ser Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile 115 120 125 Arg Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu 130 135 140 Asp Lys Phe Phe Asp Thr 145 150 <210> 84 <211> 48 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> C is Z-C <220> <221> MISC_FEATURE <222> (48)..(48) <223> L is L-benzyl-thioester <400> 84 Cys Asp Met Ala Lys Pro Asn Gly Ile Lys Val Ile Asp Asp Glu Glu 1 5 10 15 Val Lys Arg Leu Ile Arg Glu Leu Asp Ile Ala Asp Val Pro Gly Ile 20 25 30 Gly Asn Ile Thr Ala Glu Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu 35 40 45 <210> 85 <211> 78 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> R is H-R <220> <221> MISC_FEATURE <222> (78)..(78) <223> A is A-SMe <400> 85 Arg Lys Glu Val Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu 1 5 10 15 Arg Glu Tyr Ser Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr 20 25 30 Leu Asp Ile Ser Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu 35 40 45 Gly Leu Glu Ile Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val 50 55 60 Thr Val Gly Ile Ser Lys Asn Lys Val Phe Ala Lys Ile Ala 65 70 75 <210> 86 <211> 76 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> M is Ac-M <220> <221> MISC_FEATURE <222> (76)..(76) <223> M is M-OGp <400> 86 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met 65 70 75 <210> 87 <211> 198 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> C is H-C <220> <221> MISC_FEATURE <222> (198)..(198) <223> T is T-OH <400> 87 Cys Asp Met Ala Lys Pro Asn Gly Ile Lys Val Ile Asp Asp Glu Glu 1 5 10 15 Val Lys Arg Leu Ile Arg Glu Leu Asp Ile Ala Asp Val Pro Gly Ile 20 25 30 Gly Asn Ile Thr Ala Glu Lys Leu Lys Lys Leu Gly Ile Asn Lys Leu 35 40 45 Cys Asp Thr Leu Ser Ile Glu Phe Asp Lys Leu Lys Gly Met Ile Gly 50 55 60 Glu Ala Lys Ala Lys Tyr Leu Ile Ser Leu Ala Arg Asp Glu Tyr Asn 65 70 75 80 Glu Pro Ile Arg Thr Arg Val Arg Lys Ser Ile Gly Arg Ile Val Thr 85 90 95 Met Lys Arg Asn Ser Arg Asn Leu Glu Glu Ile Lys Pro Tyr Leu Phe 100 105 110 Arg Ala Ile Glu Glu Ser Tyr Tyr Lys Leu Asp Lys Arg Ile Pro Lys 115 120 125 Ala Ile His Val Val Ala Val Thr Glu Asp Leu Asp Ile Val Ser Arg 130 135 140 Gly Arg Thr Phe Pro His Gly Ile Ser Lys Glu Thr Ala Tyr Ser Glu 145 150 155 160 Ser Val Lys Leu Leu Gln Lys Ile Leu Glu Glu Asp Glu Arg Lys Ile 165 170 175 Arg Arg Ile Gly Val Arg Phe Ser Lys Phe Ile Glu Ala Ile Gly Leu 180 185 190 Asp Lys Phe Phe Asp Thr 195 <210> 88 <211> 154 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> MISC_FEATURE <223> All-D-peptide <220> <221> MISC_FEATURE <222> (1)..(1) <223> M is Ac-M <220> <221> MISC_FEATURE <222> (154)..(154) <223> A is A-SMe <400> 88 Met Ile Val Leu Phe Val Asp Phe Asp Tyr Phe Tyr Ala Gln Val Glu 1 5 10 15 Glu Val Leu Asn Pro Ser Leu Lys Gly Lys Pro Val Val Val Cys Val 20 25 30 Phe Ser Gly Arg Phe Glu Asp Ser Gly Ala Val Ala Thr Ala Asn Tyr 35 40 45 Glu Ala Arg Lys Phe Gly Val Lys Ala Gly Ile Pro Ile Val Glu Ala 50 55 60 Lys Lys Ile Leu Pro Asn Ala Val Tyr Leu Pro Met Arg Lys Glu Val 65 70 75 80 Tyr Gln Gln Val Ser Ser Arg Ile Met Asn Leu Leu Arg Glu Tyr Ser 85 90 95 Glu Lys Ile Glu Ile Ala Ser Ile Asp Glu Ala Tyr Leu Asp Ile Ser 100 105 110 Asp Lys Val Arg Asp Tyr Arg Glu Ala Tyr Asn Leu Gly Leu Glu Ile 115 120 125 Lys Asn Lys Ile Leu Glu Lys Glu Lys Ile Thr Val Thr Val Gly Ile 130 135 140 Ser Lys Asn Lys Val Phe Ala Lys Ile Ala 145 150

Claims (83)

As a method of adding at least one L-nucleotide to the 3'end of the first L-nucleic acid,
The method comprises reacting the first L-nucleic acid with the at least one L-nucleotide in the presence of a polymerase;
The polymerase may add one or more L-nucleotides to the 3'end of the first L-nucleic acid,
The polymerase is selected from the group consisting of a wild-type polymerase and a polymerase variant of the wild-type polymerase;
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15 or 1, and the amino acid of the amino acid sequence is D-amino acid;
The polymerase variant has polymerase activity and consists of an amino acid sequence, the amino acid sequence of the polymerase variant is at least one amino acid position different from the amino acid sequence of the wild-type polymerase, and the amino acid of the polymerase variant Wherein the amino acid in the sequence is a D-amino acid.
As a method of amplifying a target L-nucleic acid in the presence of L-nucleotide and polymerase,
The polymerase may amplify the target L-nucleic acid;
The polymerase is selected from the group consisting of a wild-type polymerase and a polymerase variant of the wild-type polymerase;
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15 or 1, and the amino acid of the amino acid sequence is D-amino acid;
The polymerase variant has polymerase activity and consists of an amino acid sequence, the amino acid sequence of the polymerase variant is at least one amino acid position different from the amino acid sequence of the wild-type polymerase, and the amino acid of the polymerase variant Wherein the amino acid in the sequence is a D-amino acid.
The method according to claim 1 or 2,
The method of the polymerase is a thermostable (thermostable) polymerase.
delete delete delete delete delete As a polymerase consisting of the amino acid sequence of SEQ ID NO: 15,
The amino acid of the amino acid sequence is a polymerase that is a D-amino acid.
As a polymerase consisting of the amino acid sequence of SEQ ID NO: 1,
The amino acid of the amino acid sequence is a polymerase that is a D-amino acid.
As a polymerase variant of wild-type polymerase,
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15, the polymerase variant has a polymerase activity and is composed of an amino acid sequence, and the amino acid sequence of the polymerase variant has at least one amino acid position wild-type polymerization The polymerase variant is different from the amino acid sequence of the enzyme, and the amino acid of the amino acid sequence of the polymerase variant is a D-amino acid.
As a polymerase variant of wild-type polymerase,
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 1, the polymerase variant has a polymerase activity and is composed of an amino acid sequence, and the amino acid sequence of the polymerase variant has at least one amino acid position wild-type polymerization The polymerase variant is different from the amino acid sequence of the enzyme, and the amino acid of the amino acid sequence of the polymerase variant is D-amino acid.
A composition for adding one or more L-nucleotides to the 3'end of the first L-nucleic acid, comprising a polymerase,
The polymerase is a wild-type polymerase; And a polymerase variant of the wild-type polymerase; Is selected from the group consisting of;
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15 or 1, and the amino acid of the amino acid sequence is D-amino acid;
The polymerase variant has polymerase activity and consists of an amino acid sequence, the amino acid sequence of the polymerase variant is at least one amino acid position different from the amino acid sequence of the wild-type polymerase, and the amino acid of the polymerase variant A composition in which the amino acid sequence is a D-amino acid.
A composition for amplifying a target L-nucleic acid in the presence of an L-nucleotide comprising a polymerase,
The polymerase is a wild-type polymerase; And a polymerase variant of the wild-type polymerase; Is selected from the group consisting of;
The wild-type polymerase is composed of the amino acid sequence of SEQ ID NO: 15 or 1, and the amino acid of the amino acid sequence is D-amino acid;
The polymerase variant has polymerase activity and consists of an amino acid sequence, the amino acid sequence of the polymerase variant is at least one amino acid position different from the amino acid sequence of the wild-type polymerase, and the amino acid of the polymerase variant A composition in which the amino acid sequence is a D-amino acid.
The method of claim 14,
The target L-nucleic acid amplification is a composition that is by polymerase chain reaction (PCR).
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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